cytoplasmic epigenetics inheritance by cytoplasmic

Cytoplasmic epigenetics:

inheritance by cytoplasmic continuity

Prof. Philippe Silar

Dr. Fabienne Malagnac

The screen versions of these slides have full details of copyright and acknowledgements 1

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inheritance by cytoplasmic

continuity

Philippe Silar and Fabienne Malagnac

UFR de Biochimie, Université Paris 7

Institut de Génétique et Microbiologie, Université Paris 11

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Several ways to pass information to daughter cells

- Through DNA (Avery et al., 1944)- Mendelian inheritance.- Cytoplasmic inheritance (mitochondrial DNA, plasmids and virus-like elements...).

- Through inheritable structural organization (Beisson and Sonneborn, 1964).

- Through inheritable metabolic or regulatory states (Delbrück, 1949).

* The characters are usually unstable and may "switch" between two or more forms.

* Characters are usually transmitted only through cytoplasmic

continuity.

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Structural inheritance I

Cortical inheritance in paramecium: Beisson and Sonneborn PNAS 53, 275-282

"wild-type": normal swimming behaviour "twisty": exagerated twisty swimming






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- "Twisty mutants" can be created by grafting.

- The "twisty" phenotype can be inherited over several hundreds

mitotic generations but frequently reverts to wild-type.

- The "twisty" phenotype can be inherited during meiosis:

cytotaxis

5

+

Structural inheritance II

Prion

Prion can now be transmitted without cellular continuity.

� see talk by Dr. Reed Wickner.

eRF3

aggregated[psi-][PSI+]

True & Lindquist, Nature 407, 477-483

soluble

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Struture of the chaperonin hsp60 as determined by Braig et al. (1994) Nature 371, 578-586; Braig et al.(1995) Nature Structural Biology 2,

1083-1094.

www.cryst.bbk.ac.uk

The mitochondrial chaperonin hsp60

How such an assembly machinery is itself folded and assembled?

Could it be a de novo process?

Structural inheritance III






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In-vivo assembly of newly imported wild-type hsp60 subunits

hsp60ts

23°C 37°C

W T

growth No growth

growth growth

In the mutant hsp60ts strain, the hsp60 complex fails in

folding and assembly of imported mitochondrial proteins.

hsp60ts + pGalhsp60

W T + pGalhsp60

No growth

growth

Slow growth

23°C

37°C + Gal

growth

23°C + Gal

37°C

W ild-type hsp60 complexes, assembled at permissive temperature

can rescue the mutant phenotype at restrictive temperature.

Cheng et al., Nature, 1990

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In-vitro assembly of newly imported wild-type hsp60 subunits

Assembled

hsp60

complex

23°C 37°C 23°C 37°C

100 %

25 %

50 %

75 %

WTmito

hsp60ts

mito

The correctly-folded mitochondrial chaperon in hsp60 is

required for its own assembly.

Cheng et al., Nature, 1990

9Lockshon, Genetics 2002, 161, 1425-1435

mitochondrial membrane inheritance in Saccharomyces

cerevisiae?

Structural inheritance IV

ρρρρ°[Leu+] ρρρρ° [LeuP]

Growth in medium with leucine ++ +

Growth in medium without leucine +++ +

�Unlike prions: - is semi-dominant in diploids.- is not influenced by the deletion of hsp104.

� Like prions: - does not segragate as a mendelian or mitochondrial gene.

cox4-GFP protein






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Mitochondria and more generally of organelles

ARE NOT MADE de-novo, but are produced by

growth and div ision of pre-existent structures.

Take home message

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Regulatory inheritance, the early days I

Delbrück 1949: flux equilibrium in a metabolic pathway

- There are two stable equilibrium points:

* a2 present, b2 absent.

* a2 absent, b2 present.

- There is one unstable point:

* [a2] = [b2].

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Regulatory inheritance, the early days II

Novick and Weiner (1957): The all-or-none phenomenon of the

beta-galactosidase induction

- Present at low concentration

of inducer.

- Depends on the circuitry

of the lactose operon.






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The current concept of a primary genetic

material (DNA), replicating by a template

mechanism, is opposed to a homeostatic

system operating by, perhaps, self-regulating

metabolic patterns. (Nanney 1958)

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The two basic units of the regulatory inheritance

A B

-Negative

feedbackMonostability

AB

+

A B

Positive

autoregulation

Reciprocal negative

autoregulation

Bistability

and hysteresis

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Cellular cybernetics

A B1 2

1 2

A

B

Toggle switch

Gardner et al., Nature 2000

A B1 2

1 2

A, B

Positive feedback

Becksei et al., EMBO J 2001






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What about natural systems?

Prions are commonly

understood as infectious

proteins:

Could an autocatalytic enzyme

be a prion?

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Activation of three vacuolar hydrolases in yeast:

ev idence of a phenotypic lag

Dextrose

CpY

in trans

PrA

in cis

Pro-PrAPEP4

Pro-PrB PrBPRB1

∆pep4

Pro-PrB PrB CpY

in trans

PRB1

Pro-PrA

Dextrose

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∆pep4 PRB1+/

PEP4+ PRB1+

Sporulation

Gly medium

Continuous expression of PrB supresses the loss

of down-stream hydrolase activ ities.

Roberts and Wickner, Genes & Dev. 2003

∆pep4 PRB1+

CpY+

Dex medium

Gly medium

CpY-

CpY-

∆pep4 ∆prb1

CpY-

CpY-

CpY-

∆pep4 PRB1+/

PEP4+ ∆prb1






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PrB+ cytoduct ant s

/total

PrB- PrB-

0/20

PrB+ PrB-

14/35

The stable PrB+ state can be infectious.

PrB+ = state [ββββ ] ; PrB- = state [ββββ −−−−0000 ]


∆pep4 ∆pep4

Cytoduction experiments

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[cyp-] [cyp+]

∆pep4 ∆pep4

Overexpression

of PRB1

Overexpression of PRB1 increases the frequency

of [ββββ ] appearance.


Overexpression of PRB experiment

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The cytoplasmic and infectious [ββββ ] is a prion because:

1. [ββββ ] is reversibly curable.

2. Overexpression of PRB1 increases the frequency with

which [ββββ ] appears.

But unlike “classical” prions, the [ββββ ] prion form is itself

the active form of the PRB1 gene.

Prion-like propagation can be mediated by the autocatalytic

covalent modification of an enzyme.






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Secteur-like phenomena in filamentous fungi

Nectria haematococca

Curvulariapallescens

Podospora anserina

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The basic life cycle of a filamentous fungus

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The mycelium structure

- Polarized growth.

- Cellular continuity

through septal pores.

- Cytoplasmic

exchange through cell

fusions (anastomoses).






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The development of Sectors

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The Secteur in Nectria haematococca

Stéphane Graziani, Philippe Silar and Marie-Josée Daboussi, BMC Biology 2004, 2, 18

WTWT

Mutant S* Mutant S

1

2

3

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A two-gene locus is necessary for the Secteur







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Two models


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- Greenish mycelium.- Aerial hyphae.- Fructifications (perithecia).

- Dark green mycelium.- No aerial hyphae.- No fructification.

Presented by:

-Wild-type on media rich in yeast extract.

- The PDC mutant strains.

The Crippled Growth (CG) degenerationin Podospora anserina

CG

NG

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PDC NG PDC CG

S in low

yeast extract

conditions

CG

Like prion, CG is caused by C: a cytoplasmic and infectious factor

An inducing factor is

produced in all strains even in

standard conditions.

PDC






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A system with a tamed bistability

NG CG

stationary phase

stress

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The “pink” suppressors

- Unpigmented.

- Few aerial hyphae.

- No fructification.

I II III IV V VI VII VIII IX X XI

IDC 118

172

507

404

505

343 1

308

502

506

519

508 509 511 522 510 524

C - - +- +- +- +- +- +- +- +- +-

A genetic analysis identifies genes necessary for CG

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A MAP Kinase cascade is implicated in the

production of C

IDC118, IDC507 & IDC172 = PaASK1MAPKKK

IDC404 & IDC505 = PaMKK1MAPKK

MAPKThe deletion of the PaMpk1gene results in the same phenotype.

++++

++++

CGKicka and Silar, Genetics 2004Kicka et al. in preparation






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+

1

MK*

time

Ferrell, Curr. Op. Cell. Biol. 2002, 14, 140-148

MAP kinase cascade and cellular memory

MKKK

1

MKK*MKK

MK*MK

35Bagowski & ferrel 2002, 11, 1176-1182

The active state of the

MAP kinase can be

propagated by transfer of

activated egg cytoplasm:

� this phenomenon is

analogous to the

propagation of a

cytoplasmic factor by

infection.

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Take home message

Complex regulatory interactions between different

components can generate emergent properties,

especially BISTABLE STATES, WHICH CAN BE

INHERITEDTHROUGH CELL DIVISION.






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Prion

Esterase/Lipase

Nucleus

Autocatalyticcleavage

Mitochondria

MAP kinase

module

MAP kinase

Module *

Signalingcascade

Ciliaorientation

Conclusions

- There are numerous epigenetic phenomena that occur in the

cytoplasm and that are transmitted only through cytoplasmic

continuity.

- These includes:

cytoplasmic epigenetics inheritance by cytoplasmic

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