By Hafsa Yucel

harlemchildrensociety.org

sek‐1 - Harlem Children Society

Mentor: Elyse Blum

Rockefeller University

Department of Developmental Genetics

By Hafsa Yucel


WhyistheStudyofCellDeath

Important?

Celldeathisanimportantstepinthe

developmentofanorganism

Understandingofcelldeathwillhelpuswith:



Neurodegenerativediseases

Cancerouscells,tumors


CellDeathisImportantDuring

NormalDevelopment

BaehreckeNatRevMolCellBiol(2002)


CellDeathinC.elegansisSimilarto

CellDeathinOtherOrganisms

• Caspasebasedcelldeath(apoptosis)is

presentinotherorganisms.

• Thegenesthatmediateandcontrol

programmedcelldeath(PCD)in

C.elegans:ced‐3,ced‐4,andced‐9have

similarrolesinotherorganisms.


C.elegansCellDeathPathwayis

Conserved

egl-1

BH3 domain

ced-9

ced-4

ced-3

cell death

ced-13

Bcl-2

Apaf-1

caspase


WhatisLinkerCell?

• Linkercellisthecellthatleadsandshapes

thegonadinC.elegans.(gonadisthegerm

lineandegg‐layingapparatusoftheworm.)

ANTERIOR

DORSAL

L2

Linker Cell

gonad

U.l/rp cloaca

L3/L4

L4

Adult


Linker Cell Death is Independent of all Known Cell Death

Genes

egl-1

BH3 domain

ced-9

ced-4

ced-3

cell death

ced-13

Bcl-2

Apaf-1

caspase

Abraham et al., Dev Cell (2007)


ADyingLinkerCellisNon‐apoptotic

Linker cell death morphology

Apoptotic morphology

Small white “empty” structures

Nucleus crenellated

nucleoplasm normal

Large membrane-bound vesicles

Abraham et al., Dev Cell (2007)

Images from Nature Reviews

Mol. Cell Biol. (2001) & Cell (1997)


Objective

• Tofindnewgenesthatcontrollinkercelldeath

• Totestgenestoseeiftheyactivatesek‐1or

areactivatedbysek‐1


SEK‐1FunctionsintheMAPKinase

PathwayinC.elegans

Infection/Injury

Ca ++ signaling

NSY-1

MAP3K

NSY-1

SEK-1

MAP2K

SEK-1

PMK-1

MAPK

?

Immune Response

Nerve Cell Fate

Establishment


Methodology

• Developacrossstrategytotestdifferent

genes.

• Scorenewstrainstoseeifthere’salinkercell

deathdefect.

• VisualizelinkercellbyusingGFP

*

lag-2::GFP


dpy‐5isUsedtoTrackmtk‐1

• They’rebothinchromosome1soit’seasy

totrackmtk‐1.


CrossStrategyformtk‐1

• CrossqIs56him‐5maleswithdpy‐5

hermaphrodites

• Pick6GFP+non‐dumpymales

• CrosstheGFPpositivenon–dumpymaleswith

mtk‐1hermaphrodites

• Pick18GFP+nondumpyhermaphroditesand

letthemself


CrossStrategyformtk‐1

• Scoretheplatesfortheabsenceofdumpies

• Onplateswithdumpies,pick32non‐dumpy

GFP+hermaphroditestoindividualplates,let

themself

• Lookattheplates,wewant100%GFP+and

100%non‐dumpy.


Results

• Weareatthelaststepofthecrossstrategy

rightnow.

• Thecrossissuccessfulsofarwithmtk‐1

• We’regoingtomakeanewcrosswithpek‐1

• Oncethecrossiscompleted,wewillscorefor

linkercellsurvival.


FutureWork

• Wearegoingtofinishthecrosswith

mtk‐1,andlookattheresults.

• Finishthenewcrosswithpek‐1and

qIs56him‐5totestpek‐1gene

• TestmoreMAPkinases


References

• Jacobsonetal.Programmedcelldeathin

animaldevelopment.Cell(1997)vol.88(3)pp.

347‐54

• Baehrecke.Howdeathshapeslifeduring

development.NatRevMolCellBiol(2002)vol.

3(10)pp.779‐87

• Meieretal.Apoptosisindevelopment.Nature

(2000)vol.407(6805)pp.796‐801


Acknowledgements

• Ms.ElyseBlum

• Aakanksha

• Dr.Sat

• Prof.Ross

• HCSStaff


Questions?

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