Structure/Function,Characterization,of,Kekkon5,

MQP$BIO$JBD$0021- - - - - - Structure/Function,Characterization,of,Kekkon5,

- - -

A-Major-Qualifying-Project-Report- -

Submitted-to-the-Faculty-of- -

WORCESTER-POLYTECHNIC-INSTITUTE- -

In-partial-fulfillment-of-the-requirements-for-the- -

Degree-of-Bachelor-of-Science- -

In- -

Biology-and-Biotechnology- -

By- -

___________________________________- Chloe-LaJeunesse-

-

April-27,-2017- -

- - -

APPROVED:- -

________________________-

Joseph-B.-Duffy,-Ph.-D.-

Biology-and-Biotechnology-

ABSTRACT, ,

Kekkon5- is- a- transmembrane- protein- found- in- Drosophila- melanogaster- with- an-

extracellular-region-comprised-of-leucine-rich-repeats-(LRR)-and-an-immunoglobulin-(Ig)-

domain- and- an- intracellular- region- containing- conserved- sequences- whose- functions- are-

not- well$understood.- To- further- investigate- the- significance- and- function- of- these-

intracellular-Short-Linear-Motifs-(SLiMs),-their-conservation-was-assayed-in-species-across-

the- phylum- Arthropoda- and- variants- in- which- the- second- and- third- motifs- were- missing-

from-Kekkon5-were-generated-and-assayed-in#vivo.!

ACKNOWLEDGEMENTS, , ,

,

I-would-first-like-to-thank-Duff-for-making-this-project-possible.-You-have-inspired-

me-so-much-and-have-been-a-wonderful-advisor-from-the-day-you-accepted-me-into-your-lab-

my-freshman-year.-I-will-treasure-and-carry-all-of-the-advice-and-guidance-you-have-given-

me-into-my-future-as-a-scientist.-Thank-you-for-believing-in-me.-I-would-also-like-to-thank-

Alex-Putnam-for-all-of-your-help-in-the-lab-every-day-and-for-staying-late-to-help-me-with-my-

Western-blot.-I-was-really-scared-it-wasn’t-going-to-work,-but-you-were-calm-about-it,-and-it-

ended-up-working!-Without-you,-my-days-in-lab-would-have-been-a-lot-less-fun.-Thank-you-

Natalie- Farny- for- being- a- wonderful- mentor- throughout- iGEM- and- my- undergraduate-

career-alike.--You,-too,-have-inspired-me-more-than-I-can-say-and-I-am-extremely-grateful-to-

have-had-you-as-a-guide.--And-finally,-thank-you-to-the-extraordinary-faculty-and-staff-of-the-

Department-of-Biology-and-Biotechnology.-I-have-learned-so-much-from-all-of-you-and-I-am-

truly-grateful-for-the-time-I-have-spent-here-with-you.---

TABLE,OF,CONTENTS - ,

, ,

ABSTRACT-. . . 2- -

ACKNOWLEDGMENTS-. . . 3- -

TABLE-OF-CONTENTS-. . . 4- -

INTRODUCTION . . . 5- -

MATERIALS-AND-METHODS-. . . 10- -

RESULTS-. . . 18- -

DISCUSSION-. . . -25- -

REFERENCES-. . . 28- -

APPENDIX-A:-SDM-PRIMER-SEQUENCES-AND-LOCATIONS-. . . 30- -

APPENDIX-B:-ONGOING-TRANSGENIC-F1-CROSSES-IN-DROSOPHILA . . . 31- -

APPENDIX-C:-GENE-CONSTRUCTION-CONSTRUCTS . . . . . . 32- -

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,

INTRODUCTION, -

LIG#Proteins##

-

LIGs- are- family- of- single$pass- transmembrane- proteins- that- contain- a- relatively- unique- combination- of- leucine$rich- repeats- (LRRs)- and- immunoglobulin- (Ig)- domains.- LRRs- are- a- relatively- common- motif- existing- in- hundreds- of- proteins- from- animal,- plant,- prokaryotic,-and-even-viral-species.-Functionally,-they-are-commonly-associated-with-innate- immunity,- mRNA- transport,- apoptosis,- neuronal- development,- due- to- their- ability- to- recognize- and- bind- proteins- and- other- ligands.- (Ko- &- Kim,- 2007;- Ng- et- al.,- 2010).- Structurally,-LRRs-are-comprised-of-a-core-repetitive-pattern-of-~-nine-amino-acid-resides- (L$x$x$L$x$L$x$x$N)- that- can- be- repeated- dozens- of- times- within- one- molecule.- When- stacked- together,- LRRs- adopt- a- concave,- horseshoe$like- structure.- (Padmanabhan- et- al.,- 2009).-

Like- LRRs,- Ig- domains- are- also- a- highly- common- structure.- They- are- more- characteristically-involved-in-protein-binding,-and-are-most-commonly-found-in-proteins-of- the- immune- system- such- as- antibodies- and- T$cell- receptors,- but- have- also- been- found- in- other-cell$surface-receptors,-matrix-proteins,-and-enzymes-(Bork-et-al.,-1994).-Structurally,- Ig-domains-consist-of-several-folded-antiparallel-β$sheets-connected-by-hypervariable-loop- structures- that- lend- immunoglobulin- domains- their- ability- to- bind- a- wide- range- of- structures-(Berg-et-al.,-2002).--

Though-LRRs-and-Ig$like-domains-individually-are-relatively-ubiquitous-in-proteins- throughout- many- kingdoms- of- life,- their- combination- is- quite- uncommon- and- restricted- evolutionarily.-In-the-human-proteome,-only-35-proteins-combine-LRR-and-Ig$like-domains,- compared-to-over-350-human-proteins-containing-LRRs-and-over-1100-proteins-containing- Ig$like-domains.-In-Drosophila#melanogaster,-only-nine-LIGs-exist-(Evans,-2006).-While-LIG- proteins- have- yet- to- be- widely- characterized,- past- research- has- determined- that- certain- LIGs-act-in-signaling-pathways-and-neuronal-development-(MacLaren-et-al.,-2004;-Mandai- et-al.,-2009).--

#

#

#

The#Kekkon#Subfamily#and#Kekkon#5#

#

Most- LIGs- present- in- Drosophila# melanogaster- are- members- of- the- Kekkon- (Kek)- subfamily—a-diagram-of-which-can-be-seen-in-Figure-1.-The-Kek-subfamily-is-comprised-of- six- structurally- related- proteins- (Kek1$Kek6)- that- contain- seven- LRRs- and- one- Ig$like- domain-in-their-extracellular-region-(MacLaren-et-al.,-2004).-The-cytoplasmic-domains-of- the- Kekkon- proteins- are- significantly- more- diverse,- containing- unique- but- sometimes- overlapping-combinations-of-short-stretches-of-highly-conserved-amino-acid-sequences,-or- short-linear-motifs-(SLiMs)-(MacLaren-et-al.,-2004;-Davey-et-al.,-2006).-There-are-no-direct- vertebrate- orthologs- of- the- Kek- proteins,- but- the- AMIGO/Alivin- subfamily- of- LIGS- discovered-in-vertebrates-discovered-in-2003-contain-seven-LRR-and-one-Ig$like-domain,- much- like- the- Kekkon- subfamily- (Chen- et- al.,- 2006).- While- the- structures- of- the- Kekkon- proteins-are-well$known,-the-functions-of-each-protein-are-not-as-well$understood.-While- roles-in-cellular-adhesion-and-neuronal-development-have-been-proposed-for-the-Kekkon- subfamily- in- general,- Kek1- is- the- only- member- of- the- Kekkon- subfamily- with- a- well$

characterized-function-to-date,-having-a-defined-role-in-inhibiting-Epidermal-Growth-Factor- Receptor-(EGFR)-signaling-(MacLaren-et-al.,-2004).--

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-

Figure,1:,The,Kekkon,subfamily,of,LIGs.,, ,

Structurally,- Kekkon5- is- similar- to- its- other- five- relatives,- with- an- extracellular- region-comprised-of-seven-LRRs-and-one-Ig$like-domain.-The-intracellular-region-of-Kek5-is- made-up-of-six-SLiMs-that-appear-to-be-highly-conserved—IC1,-IC2,-IC3,-IC4,-IC5,-and-IC6- (the- PDZ- binding- domain)- (MacLaren- et- al.,- 2004).- Functional- studies- of- Kek5,- have- suggested-a-role-as-a-regulator-of-bone-morphogenetic-protein-(BMP)-signaling-(Evans-et- al.,- 2009).- The- BMP- signaling- pathway- is- present- in- many- species- from- Drosophila- to- humans-and-participates-in-the-development-and-patterning-of-many-tissues.-Misregulation- of-this-pathway-can-result-in-the-development-of-kidney-disease,-hypertension,-cancer,-and- other- diseases- (Little- &- Mullins,- 2006).- In- Drosophila,- BMP- signaling- plays- a- key- role- in- dorsoventral-and-tissue-patterning-in-the-embryo-and-the-development-of-the-wing-in-the- adult- fly.- Misexpression- of- Kek5- in- the- imaginal- wing- disc,- a- structure- in- the- larvae- that- gives-rise-to-the-wing,-results-in-several-defects-associated-with-the-BMP-signaling-pathway,- such-as-the-absence-of-crossveins-in-the-wing-and-the-formation-of-ectopic-scutellar-bristles- (Evans,- 2006;- Evans- et- al.,- 2009;- Menon,- 2013).- An- example- of- these- phenotypes- can- be- seen-in-Figure-2-below.-

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Figure,2:,Missing,anterior,crossveins,in,the,wing,(top),and,ectopic,scutellar,bristle,formation, (bottom),upon,Kek5,misexpression,(Menon,,2013).,

-

Further- studies- have- shown- that- the- misexpression- of- Kek5- in- the- imaginal- wing- disc-produces-a-lethal-phenotype-in-which-cells-extrude-from-the-wing-imaginal-disc-and-die- in- Caspase$dependent- manner.- Other- phenotypes- influenced- by- Kek5- misexpression- (Figure- 3)- include- Armadillo- upregulation- at- the- adherens- junction- and- a- “large- cell”- phenotype,-in-which-Kek5$misexpressing-cells-appear-to-be-much-larger-than-normal-cells- (Menon,-2013).-

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, , , , , Figure,3:,Panels,A,and,B,represent,Armadillo,staining,in,response,to,wildtype,Kek5,(A),or, Kek5%with,deleted,intracellular,domain,(B).,Armadillo,upregulation,is,seen,in,both,cases,while,the,

“large,cell”,phenotype,is,seen,only,in,panel,A.,Panels,C,and,D,represent,light,microscope,images, showing,the,cell,extrusion,phenotype,in,response,to,wildtype,Kek5%(A),or,Kek5%with,deleted,

intracellular,domain,(D),(Menon,,2013).,

-

!

- While-the-importance-of-the-extracellular-region-of-Kek5-has-been-established,-the- role-of-the-IC-domain-remains-largely-unclear.-In-order-to-better-understand-the-role-of-the- cytoplasmic-tail,-variants-in-which-different-combinations-of-the-six-defined-IC-SLiMs-have- been-deleted-have-been-created-and-expressed-in-Drosophila-(MacLaren-et-al.,-2004;-Evans,- 2006;-Evans-et-al.,-2009;-Menon,-2013).-Misexpression-studies-with-these-variants-reveal- that-the-PDZ-binding-domain-(IC6)-is-necessary-for-Kek5-stabilization-and-localization,-and- is-thus-indispensable-for-Kek5-function-(Evans,-2006;-Evans-et-al.,-2009).-With-respect-to- the- remainder- of- the- intracellular- domain,- prior- work- indicates- it- is- critical- for- some- processes,-but-not-others-(Evans-et-al.,-2009;-Figure-3).--While-evidence-points-towards-IC1- being-dispensable-in-terms-of-overall-protein-function,-IC2,-IC3,-IC4,-and-IC5-all-appear-to-be- significant.-In-particular,-the-second-and-third-SLiMs-of-Kek5,-IC2-and-IC3,-seem-to-affect-the- formation- of- ectopic- scutellar- bristles,- the- cell- extrusion- and- death- phenotype,- and- the- large$cell-phenotype-(Menon,-2013).-However,-because-only-deletion-variants-coupling-IC2- and-IC3-with-each-other-and/or-other-SLiMs-have-been-made,-the-individual-effects-of-IC2- and-IC3-on-Kek5-function-remain-unclear.--

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% %

MATERIALS,AND,METHODS ,

#

CLUSTAL#Analysis#

Kek5- and- putative- Kek5- sequences- were- gathered- from- Drosophila# melanogaster- (fruit- fly),- Anopheles# gambiae- (mosquito),- Pediculus# humanus# corporis- (body- louse),- Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum# (flour- beetle),- Apis# mellifera- (honeybee),- two- separate- instances- of- Acromyrmex# echinatior- (new- world- ants),- and- Daphnia# pulex- (water- flea)- in- FASTA- format.- These- sequences- were- then- aligned- using- EMBL$EBI’s-(Hinxton,-Cambridgeshire)-Clustal-Omega-software.-Amino-acid-residues-were- color$coded- based- on- characteristics- such- as- hydrophobicity- and- polarity- for- easier- recognition-of-shared-properties-between-residues.--

-

Generation#of#DNA#Constructs#

The- pENTR$Kek5

- and- the- pENTRIKek5

- constructs- were- generated- synthesizing- them-from-a#pENTRIKek5-(full$length)-template-through-site$directed-mutagenesis.-Figure- 4- shows- a- summary- of- this- process,- which- was- conducted- with- NEB’s- Q5- Site$Directed- Mutagenesis-Kit.-In-two-separate-reactions-for-both-IC2-and-IC3,-8-μL-(approximately-25-ng)- of-pENTR$Kek5-template-was-combined-with-Q5-Hot-Start-High$Fidelity-2X-Master-Mixed- (12.5-μL),-distilled-water-(2-μL),-a-forward-primer,-and-a-reverse-primer-(10-μM,-1.25-μL- each)- up- to- a- final- volume- of- 25- μL.- These- primers- (Appendix- A)- were- designed- to- flank- either- IC2- or- IC3- and- read- in- opposite- directions.- In- the- case- of- Kek5

,- only- IC2- was- deleted.-In-the-case-of-Kek5

,

to-be-consistent-with-constructs-that-had-included-deletions- of-IC3-generated-in-the-past,-the-deletion-spanned-from-the-beginning-of-IC3-to-just-before- the-start-of-IC4.-The-PCR-reaction-for-both-of-these-constructs-was-run-for-25-cycles-with-an- annealing-temperature-of-62°C-and-an-extension-time-of-2-minutes-and-45-seconds.-

After- the- PCR- was- complete,- a- kinase,- ligase,- and- DpnI- (KLD)- treatment- was- prepared- for- each- reaction.- The- kinase- and- ligase- allow- the- ends- of- the- PCR- products- to- ligate- together,- re$establishing- a- circular- vector.- DpnI- is- a- restriction- enzyme- that- only- digests-methylated-DNA.-Because-the-original-pENTRIKek5#template-was-synthesized-in-E.#

coli,-the-DNA-has-been-methylated.-The-pENTR$Kek5

-and-the-pENTRIKek5

-constructs,-in-

contrast,-were-synthesized-in-a-PCR-reaction-and-thus-have-not-been-methylated.-Because- of- this,- the- addition- of- DpnI- will- degrade- the- template- DNA- while- preserving- the- newly$

synthesized- pENTR$Kek5

- or- pENTRIKek5

- constructs,- ensuring- that- the- resulting- DNA- will-have-no-template-present.--

Once- the- KLD- treatment- was- complete,- the- DNA- was- transformed- into- DH5α- cells- and-plated-onto-on-LB-agar+kanamycin-plates.-Colonies-were-picked-and-grown-up-in-5-mL- of- liquid- culture- (LB+kanamycin)- and- miniprepped- using- QIAGEN’S- QIAPREP- Spin- Miniprep-Kit.-Minipreps-of-selected-clones-were-analyzed-via-gel-electrophoresis-and-clones- likely-to-be-positive-for-pENTR$Kek5

-or-pENTRIKek5

-were-sequenced-by-Eton-Bioscience- Inc.- (Boston,- MA)- and- were- verified- in- lab- by- constructing- contigs- of- the- sequenced- fragments-of-each-construct-in-Sequencher.--

,

,

Figure,4:,Site\directed,mutagenesis,in,the,generation,of,Kek5^∆2,and,Kek5^∆3,pENTR%constructs.

-

Once-pENTR$Kek5

-or-pENTRIKek5

-were-determined-to-be-successfully-generated,- they- needed- to- be- transferred- to- an- expression- vector- by- making- use- of- the- Gateway- cloning-system.-An-overview-of-this-process-can-be-seen-in-Figure-5.---

-

-

Figure,5:,LR/BP,reactions,in,the,Gateway,cloning,system,in,relation,to,Kek5,constructs., ,

To- each- reaction- 1- μL- (approximately- 100- ng)- of- pENTR$Kek5

- or- pENTRIKek5

-

template,-1-μL-(150-ng)-of-pDEST-vector-(in-this-case,-pUASTaGFP),-6-μL-of-distilled-water,-

and-2-μL-of-Invitrogen-Gateway-LR-clonase-were-added.-The-LR-clonase-contains-integrase-

and-excisionase,-two-recombination-enzymes-from-the-lambda-bacteriophage-that-catalyze-

recombination-between-two-vectors-containing-attL-and-attR-sites.-The-attL-and-attR-sites-

become-attP-and-attB-sites,-respectively,-resulting-in-an-expression-clone-of-Kek5-deletion-

variants- flanked- by- attB- sites.- The- LR- reactions- were- left- to- incubate- at- 25°C- for- 1- hour-

before- 1- μL- (2- μg)- of- Proteinase- K- was- added- to- digest- the- recombination- enzymes- and-

terminate-the-reaction.--

Once-the-reactions-were-complete,-they-were-again-transformed-into-DH5α-cells-and- plated-onto-on-LB-agar+ampicillin-plates-to-select-for-the-expression-clone.-Colonies-were- then- picked- and- grown- in- mL- of- liquid- culture- (LB+ampicillin),- then- miniprepped- using- QIAGEN’S-QIAPREP-Spin-Miniprep-Kit.-Since-PCR-is-not-involved-in-this-cloning-step-there-is- little-opportunity-for-replication-errors-or-mutation-in-the-LR-reaction-process.-Therefore,- only-reactions-for-the-∆2-and-∆3-junctions-and-the-GFP-region-of-each-construct-were-sent- out- for- sequencing- by- Eton- Bioscience- Inc.- (Boston,- MA).- Because- the- original- Kek5- sequence- did- not- include- a- stop- codon,- when- translated,- the- Kek5- ∆2- and- ∆3- deletion- variants-will-be-tagged-with-the-eGFP-sequence-included-in-the-original-pUASTaGFP-vector,- which-does-include-a-stop-codon.-

Upon-verification-of-the-correct-Kek5

-or-Kek5

-inserts-in-pUASTaGFP,#midipreps-of- each- constructs- were- prepared- with- QIAGEN’S- QIAGEN- Plasmid- Midi- Kit- to- ensure- that- enough-DNA-would-be-generated-to-be-sent-off-for-injection-into-Drosophila#melanogaster- embryos.,

#

Gal4/UAS#System#

The-Gal4/UAS-system-is-a-gene-regulatory-system-used-in-research-for-conducting-

gain-of-function-studies-through-controlled-gene-expression-(Duffy,-2002).-The-Gal4-gene-

encodes- a- protein- capable- of- regulating- the- transcription- of- genes- associated- with- an-

Upstream- Activating- Sequences- (UAS)- by- directly- binding- to- the- UAS- to- activate-

transcription.-Originally-discovered-in-the-yeast-Saccharomyces#cerevisiae,-this-system-has-

been- adapted- for- use- in- many- organisms.- For- organismal- gain- of- function- studies- in-

Drosophila,# this- system- is- employed- through- the- crossing- of- driver- (Gal4)- and- responder-

(UAS)- lines.- Lines- of- transgenic- flies- called- responders- that- contain- the- gene- of- interest-

associated-with-UAS-elements-possess-this-gene,-but-do-not-express-it,-as-no-Gal4-is-present-

to- activate- transcription.- These- responders- are- mated- to- lines- of- flies- expressing- Gal4- in-

different-tissues-and-patterns,-the-drivers.-This-results-in-progeny-expressing-the-gene-of-

interest- in- the- tissues- associated- with- the- expression- of- Gal4- in- the- driver- lines- (Duffy,-

2002).- An- overview- of- this- system- is- featured- in- Figure- 6.- This- system- allows- for- the-

misexpression- of- genes- in- Drosophila# (and- other- eukaryotes)- while- also- allowing- for- the-

Gal4/UAS- system- is- also- used- for- gain- of- function- studies- through- the- co$transfection- of- driver-and-responder-sequences-directly-into-the-culture-(Klueg-et-al.-2002).-The-drivers- used- in- these- cases- can- be- constitutively- expressed,- such- as- the- ArmIGal4# driver,- or- inducible,- such- as- the- mtIGal4# driver,- for- which- expression- can- be- induced- by- the- introduction-of-copper-or-other-heavy-metals-(Klueg,-et-al.,-2002).--

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#

#

#

#

#

#

#

#

#

#

#

#

Figure,6:,The,Gal4,misexpression,system,in,Drosophila%melanogaster,(Duffy,,2002).

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#

Generation#of#Transgenics#

50-μG-each-of-pUASTaKek5

GFP-and-pUASTaKek5

GFP#DNA-was-sent-to-Best-Gene,- Inc.- (Chino- Hills,- CA)- for- injection- into- w

- embryos.- After- injection,- the- larvae- were- mailed- back- to- Duffy- lab- for- transgenic- screening.- Once- the- larvae- eclosed,- the- resulting- flies-(generation-G

)-were-single$pair-mated-to-2$3-w

-males-or-females.-The-pUASTaGFP- vector- the- Kek5

- and- Kek5

- genes- were- transposed- into- contained- a- copy- of- the- white- gene-under-its-own-set-of-regulatory-elements.-Therefore,-any-F

-progeny-from-the-G

-cross- with- eye- color- are- likely- to- contain- the- transgene.- The- F

offspring- of- the- G

- cross- were- screened- for- eye- color,- and- any- putative- transgenics- gathered- from- separate- G

- crosses- were- considered- independent- insertion- events.- Putative- transgenic- offspring- (generation-

!

F

)- for- each- line- were- single$pair- mated- to- 2$3- w

- males- or- females.- This- mating- of- putative- transgenics- to- w

ensures- the- amplification- of- all- transgenic- lines- for- stable- stock-generation-and-future-gene-mapping.-

-

Transfection#in#Cell#Culture##

# To- confirm- Kek5

GFP- and- Kek5

GFP- expression- prior- to- the- generation- of- the- transgenics,-pUASTaKek5

GFP-and-pUASTaKek5

GFP#were-transfected-into-S3-cells,-a-cell- line- derived- from- male- Drosophila# melanogaster- embryonic- cells.- First,- each- DNA- sample- was-purified-using-a-phenol:chloroform-extraction.-An-equal-volume-of-phenol:chloroform- was- added- to- a- 20- μL- aliquot- of- each- sample- and- after- vortexting,- the- samples- were- centrifuged-for-2-minutes-at-room-temperature.-To-each-tube,-5-M-NaCl-(in-a-ratio-of-1:20)- and-100%-EtOH-(in-a-ratio-of-2:1)-were-added-to-precipitate-the-DNA.-The-solutions-were- incubated-on-ice-for-10-minutes,-then-centrifuged-to-pellet-the-DNA.-Then,-the-DNA-pellet- was-washed-with-70%-EtOH,-centrifuged-again,-decanted,-and-left-to-air-dry.-Once-dry,-the- pellets-were-taken-up-in-TE-buffer.-The-concentration-of-each-sample-was-measured-on-a- NanoDrop,-and-each-construct-was-diluted-to-a-concentration-of-100-ng/µL.-

- Prior- to- transfection,- S3- cells- were- split- into- a- T75- flask- maintained- at- normal- growth-conditions-(25°C,-no-CO

).-One-day-before-transfection,-on-the-day-the-cells-would- normally- be- split,- the- S3- cells- in- the- T75- were- resuspended- in- Schneider’s- Media- and- transferred-to-a-15-mL-conical-tube.-An-aliquot-of-cells-was-taken-from-this-tube,-diluted- 1:10,-and-counted-on-a-hemocytometer.-Then,-according-to-the-appropriate-dilution-factor,- 5-x-10

-cells-were-seeded-into-each-well-of-a-6-well-cell-culture-plate-at-a-final-volume-of-1.6- mL-per-well.-The-plates-were-incubated-at-normal-growth-conditions-for-24-hours-until-a- confluence-of-approximately-80$100%-was-reached.-

- The-next-day,-four-wells-were-co$transfected-with-400-ng-of-ArmIGal4-(a-constitutive-

driver)- and- 400- ng- of- either- pUASTaKek5

GFP,# pUASTaKek5

GFP,- pUASTaKek5

GFP,- or-

nothing-(negative-control)-in-EC-buffer.-Once-Enhancer-and-Effectine-Transfection-reagent-

were-added-and-incubated-in-sequence,-the-media-of-each-well-was-aspirated-and-each-well-

was-washed-with-1X-PBS,-which-was-then-aspirated,-as-well.-1.6-mL-and-0.6-mL-of-fresh-

Schneider’s-Media-was-added-to-each-well-and-each-co$transfection-mix,-respectively.-Each-

co$transfection-mix-was-added-to-an-appropriately-labelled-well-and-the-plates-were-left-to- incubate-at-normal-growth-conditions-overnight.-

#

GFP#Imaging-

Because- the- Kek5# constructs- were- tagged- with- GFP,- the- wild- type- and- variant- proteins-produced-should-be-visible-through-fluorescent-microscopy.-In-order-to-visualize- the-Kek5$GFP-proteins-and-observe-expression-and-localization-patterns,-the-co$transfected- plate- was- viewed- under- under- Zeiss- SteREO- Discovery.V12- microscope- at- 100X- either- at- 480- nm- (GFP- fluorescence)- or- under- incandescent- light- (cell- images).- Images- were- captured-with-a-monochrome-Zeiss-Axiocam-and-processed-with-the-Axiovision-software.-- -

Trypan#Blue#Vital#Stain##

At-day-6-post$transfection,-an-aliquot-of-100-μL-of-resuspended-cells-was-taken-from- each-co$transfection-well-and-combined-with-100-μL-of-Trypan-blue,-which-is-a-stain-only- taken- up- by- dead- cells.- For- each- co$transfection,- 10- μL- of- this- Trypan- blue- mixture- was- loaded-into-a-hemocytometer-and-the-numbers-of-living-and-dead-cells-were-counted-in-two- separate-squares-and-totaled.-The-percentage-of-dead-cells-to-the-total-number-of-cells-for- each-co$transfection-was-then-calculated.-

#

Western#Blot#

In-order-to-detect-potential-Kek5

GFP,-Kek5

GFP,-and-Kek5

GFP-expression,-125- μL-(approximately-2-x-10

-cells)-of-each-co$transfection-was-aliquoted,-spun-down,-taken- up- in- 1- mL- of- sample- buffer- (12- mM- TrisCl,- 5%- glycerol,- 0.4%- SDS,- 2.9- mM- βME,- 0.02%- bromophenol-blue),-and-stored-at-$20°C.-Just-before-loading,-two-aliquots-of-5-μL-and-20-μL- for- each- sample- were- made- and- lysed- by- boiling- for- 5- minutes.- Samples- were- run- on- precast- 7.5%- polyacrylamide- gels- from- BIORAD- at- 20mA- for- 1- hour.- Proteins- were- then- transferred-from-the-gel-to-a-nitrocellulose-membrane-100V-for-1-hour.-In-order-to-confirm- the- transfer,- the- membrane- was- Ponceau- stained,- then- blocked- for- 1- hour.- Then,- the- membrane-was-blocked-in-5%-NFDM-in-TBST-(100-mM-Tris,-150-mM-NaCl,-0.1%-Tween$

20)- for- 1- hour,- after- which- the- membrane- was- rinsed- and- incubated- with- the- primary-

antibody-(monoclonal-α$GFP)-diluted-in-0.5%-NFDM-in-TBST-overnight-at-4°C.--

The-next-day,-the-membrane-was-washed-in-TBST-and-incubated-with-the-secondary- antibody- (goat- α$mouse,- diluted- 1:20,000- in- 5%- NFDM- in- TBST)- for- one- hour- at- room- temperature.- The- membrane- was- once- again- washed- in- TBST- and- incubated- in- HRP- substrate-for-10-minutes,-then-photographed-with-chemiluminescent-imaging.,

-

% %

RESULTS#

#

There- is- much- evidence- to- support- links- between- the- IC- domain- of- Kek5- and- its- overall- function.- Phylogenetic- analysis- reveals- that- all- SLiMs- found- in- the- IC- domain- of- Kek5-show-a-relatively-high-level-of-conservation-in-closely-related-species,-suggesting-an- evolutionary-advantage-to-maintaining-these-sequences-(MacLaren-et-al.,-2004;-Talwar-and- Duffy,- personal- communication).- Additionally,- past- research- has- shown- that- both- the- IC- domain-as-a-whole-and-certain-combinations-of-IC-SLiMs-have-large-effects-on-the-function- of-Kek5-(Evans,-2006;-Menon,-2013).-Among-these-combinations,-IC2-and-IC3-in-particular- have- been- shown- to- have- a- significant- effect- on- phenotypes- associated- with- Kek5- misexpression-when-deleted-alongside-IC1.-However,-variants-in-which-IC2-and-IC3-have- been- individually- deleted- have- not- yet- been- successfully- generated.- Therefore,- a- further- investigation- on- the- both- the- significance- of- these- domains- and- their- individual- contributions-to-Kek5-function-was-undertaken.--

#

Phylogenetic#Analysis#of#IC2#and#IC3#Conservation#in#Kekkon5#Homologs-

An- alignment- of- Kek5- orthologs- and- putative- orthologs- was- created- using- Clustal- Omega.-A-sampling-of-Kek5-protein-sequences-for-closely$related-and-less-closely$related- members- of- Hexapoda- were- analyzed- as- well- as- one- member- of- Crustacea.- The- species- sampled- for- this- alignment- were,- in- order- of- sequence- in- Figures- 7- and- 8,- Daphnia# pulex- (water-flea),-Anopheles#gambiae-(mosquito),-Drosophila#melanogaster-(fruit-fly),-Pediculus#

humanus# corporis- (body- louse),- Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum#

(flour- beetle),- Apis# mellifera- (honeybee),- and- two- separate- instances- of- Acromyrmex#

echinatior-(new-world-ants).--

Figure- 7- shows- the- portion- of- the- alignment- featuring- the- SLiM- $- IC2- (fingerprint- PDLL).- The- alignment- of- IC2- reveals- that- sequences- highly- similar- to- Drosophila#

melanogaster’s-Kek5-IC2-exist-in-all-sampled-sequences.- -

-

-

Figure,7:,Clustal,Omega,alignments,of,Kek5,IC2,in,representative,species,of,Crustacea,(Dpulk5/6),, and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,

-

Likewise,- Figure- 8- shows- the- portion- of- the- alignment- featuring- SLiM- $- IC3- (fingerprint-TIPR).-This-alignment-found-that-sequences-closely-related-in-sequence-to-the- Kek5-IC3-region-of-Drosophila#melanogaster-exist-in-all-of-the-aligned-sequences.-,

-

-

Figure,8:,Clustal,Omega,alignments,of,Kek5,IC3,in,representative,species,of,Crustacea,(Dpulk5/6),, and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,,

-

#

Design#of#Novel#Kek5

!and$Kek5

!Constructs#

Previously,-four-variants-of-Kek5-in-which-IC2-and-IC3-were-deleted-in-combination-

with-other-SLiMs-have-been-generated.-These-constructs-yielded-high-rates-of-lethality-and-

additional- phenotypic- effects- (i.e.- extrusion,- cell- death,- large- cell- phenotype)- when-

expressed- in- Drosophila# melanogaster- (Menon,- 2013).- To- form- a- better- idea- of- the-

individual-effects-and-contributions-of-IC2-and-IC3,-constructs-of-Kek5

-and-Kek5

-were-

first-synthesized-in-a-pENTR-vector-via-site$directed-mutagenesis-of-an-existing-full-length-

pENTRIKek5-construct.-Then,-Kek5

-and-Kek5

-were-shuttled-from-the-entry-vector-into-

the- expression- vector- pUASTaGFP.- Figure- 9- shows- the- structure- of- the- Kek5- gene- for- wildtype,-Kek5

,-and-Kek5

#with-their-respective-lengths.-

-

-

Figure,9:,Wildtype,Kek5,and,novel,Kek5,variants:,Kek5^∆2,and,Kek5^∆3.,

#

Both-the-pENTR-and-pUASTaGFP-constructs-for-each-deletion-variant-were-sequence- verified.-The-pENTR#constructs-for-Kek5

-and-Kek5

-were-fully-sequenced-from-the-start- codon-through-the-end-of-the-gene-to-verify-that-no-mutations-had-accumulated-during-the- polymerase- chain- reaction.- Because- there- is- little- risk- of- mutation- upon- shuttling- a- gene- from- one- P$element- vector- to- another- via- the- Gateway- cloning- system,- only- the- deletion- junction- and- terminal- region- of- pUASTaKek5

GFP- and- pUASTaKek5

GFP- needed- to- be- sequenced-to-sufficiently-verify-the-final-constructs.-

-

Generation#of#Transgenic#Lines-

Larvae- injected- with- pUASTaKek5

GFP- and- pUASTaKek5

GFP# constructs- were- received-after-mailing-DNA-off-for-injection.-In-total,-143-adult-flies-(Generation-G

)-were- received- from- these- injections—48- injected- with- pUASTaKek5

GFP# and- 95- injected- with- pUASTaKek5

GFP.#Each-of-these-flies-were-single$pair-mated-to-2$3-W

-males-or-females.- From-these-crosses,-7-putative-Kek5

#and-9#putative#Kek5

#F

transgenics-were-collected.- These-putative-transgenics-have-been-crossed-to-2$3-W

-males-or-females,-depending-on- the-sex-of-the-transgenic.--

Figure-10-shows-a-summary-of-the-overall-plan-and-current-state-of-the-generation- of-transgenic-Kek5

#and-Kek5

#lines.-These-lines-have-yet-to-be-mapped-and-stabilized,-but-

LRR Ig Tm 1 2 3 4 5

LRR Ig Tm 1 3 4 5

LRR Ig Tm 1 2 4 5

Kek5 wt 2751 bp

Kek5

2664 bp

Kek5

2580 bp

the- process- of- doing- so- for- each- line- has- already- begun.- After- the- lines- are- mapped- and- stable-stocks-are-produced,-functional-tests-can-be-begin-by-crossing-the-Kek5

#and-Kek5

# lines-to-lines-expressing-a-Gal4-driver.--

-

Figure,10:,Flowchart,of,the,plan,and,progress,for,generating,and,testing,stable,stocks,of,Kek5^∆2,and, Kek5^∆3,deletion,variants,in,Drosophila%melanogaster.%,

-- -

Expression#and#Localization#of#Kek5#Variants#in#Cell#Culture#

- In- order- to- confirm- the- expression- and- localization- of- the- two- deletion- variants,- pUASTaKek5

GFP-and-pUASTaKek5

GFP,-these-constructs-were-co$transfected-into-an-S3- cell- line- with- a- constitutive- ArmIGal4- driver.- At- 2,- 5,- and- 6- days- post$transfection,- fluorescent- and- bright- field- microscopy- images- were- taken- of- each- of- the- two- deletion- variant- constructs- and- positive- (ArmIGal4- +- pUASaKek5

GFP)- and- negative- (ArmIGal4- alone)-transfection-controls.-These-images-have-been-compiled-into-Figure-11-below.--

Generate Constructs (2)

Functional Tests Design Variants

Create Transgenics

(48 Kek5

crosses, 95 Kek5

crosses)

Map & Create Stable Stocks (all lines)

Localization/Expression Studies (GFP-based)

Misexpression Studies

(A9-Gal4 & Ptc-Gal4 by all lines)

-

Figure,11:,Fluorescent,microscopy,of,Arm9Gal4,+,UAS9Kek5^wtGFP,,UAS9Kek5^∆2GFP,,and,UAS9Kek5^∆3GFP, co\transfections,and,Arm9Gal4,control,on,day,2,,day,5,,and,day,6,after,transfection.,Light,microscope,

images,are,displayed,in,each,corner,inset.,

-

- On-day-2-post$transfection,-the-UASIKek5

GFP,#UASIKek5

GFP,-and-UASIKek5

GFP- ArmIGal4-co$transfections-all-display-GFP-that-appears-to-be-localized-to-the-membrane-of- each-cell-consistent-with-the-localization-and-expression-pattern-of-Kek5.-As-expected,-the- control,-which-has-been-transfected-with-only-the-ArmIGal4-driver,-does-not-display-GFP.- On-day-5-post$transfection,-the-UASIKek5

GFP-and-UASIKek5

GFP-transfections-display-a- similar- pattern- with- an- apparent- increase- in- levels- and- proportion- of- cells.- However,- surprisingly-starting-at-day-5-post$transfection,-the-UASIKek5

GFP-co$transfection-ceases- to-display-a-GFP-signal.-This-same-pattern-is-seen-on-day-6-post$transfection.-

#

Cytotoxic#Effect#of#Kek5

#

The- data- gathered- from- the- GFP- localization- and- expression- analysis- in- the- transfections-revealed-a-decrease-in-GFP-expression-starting-at-day-5-post$transfection-for- the-ArmIGal4-+-UASIKek5

GFP-co$transfection.-Based-on-this-data,-it-was-hypothesized-that- the-cells-in-the-ArmIGal4-+-UASIKek5

GFP#co$transfection-were-dying.-In-order-to-test-this,- a-vital-stain-(trypan-blue)-was-conducted-using-aliquots-of-cells-from-each-transfection-at- day- 6- post$transfection.- - A- bar- graph- of- the- percentages- of- dead- cells- observed- for- each- transfection-can-be-seen-in-Figure-12.--

-

,

Figure,12:,Percentage,of,dead,cells,in,trypan,blue,cell,counts,of,Arm\Gal4,+,UAS\Kek5^wtGFP,,UAS\

Kek5^∆2GFP,,and,UAS\Kek5^∆3GFP,co\transfections,and,Arm\Gal4,control,at,day,6,after,transfection., 0%

10%

20%

30%

40%

50%

60%

UAS Kek5wt GFP! UAS Kek5Δ2 GFP!

UAS Kek5Δ3 GFP!

Arm-Gal4!

% ce ll d e a th !

Effect of IC Variants on Cell Viability!

The- counts- of- dead- versus- living- cells- in- each- co$transfection- aliquot- reveal- a- percentage-of-cell-death- that- is-dramatically-increased-in-the-UASIKek5

GFP-transfection- relative-to-the-other-three-transfections.-The-percentage-of-dead-cells-observed-in-the-UASI Kek5

GFP,-UASIKek5

GFP,-and-the-Arm$Gal4-transfections-is-consistently-close-to-or-below- 10%.- In- contrast,- the- percentage- of- dead- cells- seen- in- the- UASIKek5

GFP- transfection- counts-is-nearly-50%,-suggesting-a-higher-rate-of-death-in-cells-expressing-Kek5

GFP.- -

Analysis#of#Kek5#Variant#Protein#Expression-

In-another-attempt-to-confirm-the-expression-of-Kek5

GFP-and-Kek5

GFP,-an-anti$

GFP-western-blot-was-run-with-cell-lysates-at-day-6-post$transfection-from-each-of-the-four- co$transfections.- Figure- 13- is- an- image- of- the- resulting- blot- with- lanes- labelled- and- the- expected-sizes-Kek5

GFP-and-UAS$Kek5

GFP-listed.--

,

Figure,13:,Anti\GFP,Western,blots,of,Arm\Gal4,+,UAS\Kek5^wtGFP,,UAS\Kek5^∆2GFP,,and,UAS\Kek5^∆3GFP, co\transfections.,

-

Western-blot-showed-that-Kek5

GFP-(lane-1)-is-running-close-to-its-expected-size- (130-kDa)-and-that-UAS$Kek5

GFP-(lane-3)-is-running-slightly-below-at-what-seems-to-be- its-expected-size-(124-kDa).-UAS$Kek5

GFP-did-not-appear-on-the-western-blot.-However,- given- that- these- cell- lysates- were- taken- at- day- 6- post$transfection,- a- time- point- at- cells- expressing- UAS$Kek5∆2GFP- were- undergoing- death- and- had- already- ceased- giving- off- a- GFP-signal,-the-absence-of-a-signal-for-the-∆2-variant-was-expected.-

,, ,

UAS -Ke

k5

Δ3 GF P

UAS -Ke

k5

Δ2 GF P

UAS -Ke

k5

wt GF

P

124 kDa 130-kDa

DISCUSSION,

#

If- cells- cannot- communicate- with- one- another,- than- a- cohesive- multicellular- organism- cannot- develop.- Cell- signaling- is- a- crucial- aspect- of- the- development- of- all- animals,-and-while-there-are-many-pathways-throughout-all-existing-species,-many-of-these- pathways- are- highly- conserved.- One- such- highly$conserved- pathway- is- the- Bone- Morphogenetic-Protein-(BMP)-pathway,-which-influences-the-patterning-and-development- of- many- different- types- of- tissues- from- Drosophila# to- humans.- Dysregulation- of- the- BMP- pathway- can- result- in- the- generation- of- tumors- and- other- diseases- in- humans- (Little- &- Mullins,-2006).-Because-the-BMP-signaling-pathway-is-widely-conserved,-the-more-insight- that-can-be-gathered-on-this-pathway-and-how-it-is-regulated,-the-better.--

In- the- past,- the- intracellular- region- of- Kek5- has- been- demonstrated- to- play- some- role-in-regulating-BMP-signaling-and-to-influence-certain-lethal-cell-phenotypes.-The-second- and-third-SLiMs-of-the-intercellular-region-of-Kek5-in-particular,-IC2-and-IC3,-seem-to-be- important-in-regulating-some-aspects-of-BMP-signaling,-as-their-deletion-in-misexpression- studies-seems-to-influence-a-higher-rate-of-scutellar-bristle-duplication-in-adult-flies-and-IC2- and-IC3-have-also-been-shown-to-be-key-in-the-development-of-the-lethal-cell-extrusion-and- large- cell- phenotypes- observed- upon- Kek5- misexpression- in- the- 3

- instar- wing- imaginal- disc-(Menon,-2013).-Despite-their-apparent-significance-and-the-large-array-of-Kek5-IC-SLiM- deletion-variants-that-have-been-generated-in-the-past,-their-respective-roles-are-unknown,- as-individual-deletion-variants-have-not-yet-been-generated.-Therefore,-in-order-to-allow-for- the-determination-the-significance-and-specific-functions-of-IC2-and-IC3,-a-small-analysis-of- IC2- and- IC3- conservation- was- undertaken- and- individual- deletion- variants,- Kek5

- and- Kek5

,-were-generated.-

- The-results-of-the-phylogenetic-analysis-revealed-that,-within-Hexapoda,-sequences- representative-of-IC2-and-IC3-are-highly-conserved-within-Kek5-homologs.-Even-in-Daphnia#

pulex,-a-member-of-Crustacea-separated-evolutionarily-from-Hexapoda-by-millions-of-years,-

IC2-and-IC3-are-still-present-within-this-species’-homolog-of-Kek5.-This-large-time-span-of-

conservation-suggests-that,-whatever-their-function-may-be,-IC2-and-IC3-are-indispensable-

for-Kek5-function.-However,-while-Kek5-orthologs-were-searched-for-in-other-members-of-

Crustacea- and- members- of- Chelicerata,- there- is- not- much- data- on- orthologous- protein- sequences-in-general-for-these-species.-In-the-future,-this-study-may-produce-results-more- representative- of- Kek5- IC2- and- IC3- conservation- in- Arthropoda- species- outside- of- Hexapoda.--

- Based- on- the- sequencing- information- for- each- construct,- and- the- expression- data,- the- generation- of- expressible- Kek5

# and# Kek5

constructs- was- successful.- While- co$

transfected- cells- expressing- Kek5

- exhibited- increased- death- by- day- 5- post$transfection,- the-microscopy-images-taken-on-day-2-post$transfection-suggest-that-Kek5

$GFP-is-being- expressed- and- is- localizing- to- the- cell- membrane- much- like- Kek5

$GFP- and- Kek5

$GFP.- However,-a-western-blot-of-Kek5

$GFP-at-earlier-times-points-have-yet-to-be-performed.-In- a- future- experiment,- a- new- co$transfection- of- ArmIGal4# and- pUASTaKek5

GFP- should- be- set- up,- and- the- cells- from- that- co$transfection- should- be- harvested- around- day- 2- post$

transfection-for-use-in-a-Western-blot-to-confirm-expression-and-the-size-of-this-protein.-At- the- same- time,- a- more- closely- monitored- time- course- should- be- taken- of- the- Kek5

- co$

transfection-to-gather-more-information-on-the-transition-from-cells-expressing-Kek5

$GFP- to-cells-dying-off.-In-this-way,-it-can-be-determined-whether-or-not-the-observed-cell-death-is- linked-to-the-expression-of-Kek5

$GFP-and-how-this-cell-death-is-occurring.--

- However,- while- Kek5

$GFP- could- not- be- visualized- on- a- Western- blot- due- to- cell-

death- and- subsequent- protein- degredation,- the- cell- death- seen- upon- expression- of- this-

protein-is-a-highly-interesting-result.-The-stark-contrast-between-the-high-rates-of-cell-death-

seen- in- the- UASIKek5

IGFP- co$transfection- and- the- healthy- cells- seen- in- the- UASIKek5

I

GFP# co$transfection- provides- the- first- set- of- evidence- for- IC2- and- IC3- having- distinct-

functions- from- one- another.- Based- on- the- increased- cell- death- seen- in- cells- expressing-

Kek5

$GFP,-IC2-seems-to-be-more-crucial-to-Kek5-function,-and-the-previously-suggested-

notion-that-this-region-is-a-regulator-of-Kek5-itself-is-supported-by-the-data-presented-here.-

- The- generation- of- stable- stocks- of- pUASTaKek5

GFP# and# pUASTaKek5

GFP# is- a-

crucial-aspect-of-determining-IC2-and-IC3-function-in#vivo.-So-far,-F

-transgenics-have-been-

successfully- gathered- from- the- G

- crosses- set- up- with- w

- from- the- original- injected-

larvae.-These-F

-transgenics,-signified-by-the-presence-of-eye- color,-have-been-crossed-to-

w

- and- the- lab- is- still- awaiting- the- F

- generation- from- these- crosses.- Once- the- F

-

generation-is-produced,-the-chromosomal-location-of-the-Kek5

-or-Kek5

-gene-insertion-

can-be-mapped-and-stable-stocks-can-be-generated.-These-stable-stocks-can-be-crossed-to- A9.Gal4- or- Ptc.Gal4- flies- to- initiate- Kek5

- or- Kek5

- expression- in- the- offspring- of- such- a- cross.- While- this- means- that- misexpression- studies- for- Kek5

- or- Kek5

- in- Drosophila#

melanogaster-are-still-a-few-months-off,-the-crucial-first-steps-in-this-process-have-already- been-taken.-

In-past-studies,-cell-culture-has-not-been-used-as-a-tool-for-observing-misexpression- effects- of- Kekkon5- and- its- many- deletion- variants.- This- study- has- shown- that- co$

transfection- of- UASIKek5IGFP# constructs- with- a- Gal4- driver- into- Drosophila# cells- can- provide- a- relatively- quick- and- easy- method- for- assessing- some- functionalities- associated- with-SLiMs-of-Kek5,-especially-compared-to-the-long-process-of-generating-stable-transgenic- lines-in-Drosophila#melanogaster.-

- With-Kek5-playing-a-regulatory-role-in-BMP-signaling,-a-pathway-conserved-in- vertebrates-that-influences-the-development-of-healthy-tissues-and-cancerous-cells-alike,- the-characterization-of-Kek5-has-implications-for-combating-BMP$related-diseases-more- effectively-and-better-understanding-vertebrate-development.-With-constructs-deleting-IC2- and-IC3-of-Kek5-successfully-generated,-and-a-cell-death-activity-correlated-with-the-

deletion-of-the-SLiM-$-IC2,-a-key-link-between-the-intracellular-structure-and-sequence-of- Kek5-to-it’s-function-has-been-elucidated.--With-the-extensive-array-of-intracellular-variants- and-tools-to-assess-cell-death-pathways-readily-available,-rapid-progress-on-defining-the- mechanism-underlying-the-role-of-IC2,-and-ultimately-Kek5,-in-cell-death/viability-can-now- be-made.-

-

REFERENCES , -

Berg-J.M.,-Tymoczko,-J.L.,-and-Stryer-L.-(2002).-Biochemistry.-New-York:-W.-H.-Freeman-and- Company.-

Bork,-P.,-Holm-L.-and-Sander,-C.-(1994).-The-Immunoglobulin-Fold:-Structural-Classification,- Sequence-Patterns-and-Common-Core.-J.#Mol.#Biol.#242,-309$320.-

Chen,-Y.,-Aulia,-S.,-Li,-L.-and-Tang,-B.L.-(2006).-AMIGO-And-Friends:-An-Emerging-Family-of- Brain$Enriched,-Neuronal-Growth-Modulating,-Type-I-Transmembrane-Proteins-with- Leucine$Rich-repeats-(LRR)-and-Cell-Adhesion-Molecule-Motifs.-Brain#Res.#Rev.#51,-265$274.- Davey,-N.E.,-Sheilds,-D.C.,-and-Edwards,-R.J.-(2006).-SLiMDisc:-Short,-Linear-Motif-Discovery,- Correcting-for-Common-Evolutionary-Descent.-Nucleic#Acids#Res.#34,-3546$3554.--

Duffy,-J.B.-(2002).-GAL4-System-in-Drosophila:-A-Fly-Geneticist’s-Swiss-Army-Knife.-Genesis#

34,-1$15.-

Evans,-T.-(2006).-Characterization-of-Kekkon5,-a-Drosophila#LIG-Protein-that-Modulates- BMP-and-Integrin-Function.-PhD-thesis.-Indiana-University.-

Evans,-T.,-Haridas,-H.,-and-Duffy,-J.B.-(2009).-Kekkon5-is-an-Extracellular-Regulator-of-BMP- Signaling.-Dev.#Biol.-326,-36$46.-

Klueg,-K.M.,-Alvarado,-D.,-Muskavitch,-M.A.T.,-Duffy,-J.B.-(2002).-Creation-of-a-GAL4/UAS$

Coupled-Inducible-Gene-Expression-System-for-Use-in-Drosophila-Cultured-Cell-Lines.- Genesis#34,-119–122.-

Ko,-J.-and-Kim,-E.-(2007).-Leucine$Rich-Repeat-Proteins-of-Synapses.-J.#Neurosci.#Res.#85,- 2824$2832.-

Little,-S.-and-Mullins,-M.-(2006).-Extracellular-Modulation-of-BMP-Activity-in-Patterning-the- Dorsoventral-Axis.-Birth#Defects#Res.,#Part#C#78,-224$242.-

MacLaren,-C.M.,-Evans,-T.A.,-Alvarado,-D.,-and-Duffy,-J.B.-(2004).-Comparative-Analysis-of-the- Kekkon-Molecules,-Related-Members-of-the-LIG-Superfamily.-Dev.#Genes#Evol.#214,-360$366.- Mandai,-K.,-Guo,-T.,-St-Hillaire,-C.,-Meabon,-J.S.,-Kanning,-K.C.,-Bothwell,-M.,-and-Ginty,-D.D.- (2009).-LIG-Family-Receptor-Tyrosine-Kinase$Associated-Proteins-Modulate-Growth-Factor- Signals-during-Neural-Development.-Neuron#63,-614$627.-

Menon,-H.-(2013).-Deciphering-the-Role-of-Kekkon5-in-BMP-Signaling-and-Cell-Junction-

Biology.-PhD-thesis.-Worcester-Polytechnic-Institute.--

Musacchio,-M.,-and-Perrimon,-N.-(1996).-The-Drosophila-Kekkon-Genes:-Novel-Members-of- Both-the-Leucine$Rich-Repeat-and-Immunoglobulin-Superfamilies-Expressed-in-the-CNS.- Dev.#Biol.#178,-63$76.-

Ng,-A.C.,-Eisenberg,-J.M.,-Heath,-R.J.,-Huett,-A.,-Robinson,-C.M.,-Nau,-G.J.,-and-Xavier,-R.J.- (2010).-Human-Leucine$Rich-Repeat-Proteins:-A-Genome$Wide-Bioinformatic-

Categorization-and-Functional-Analysis-in-Innate-Immunity.-Proc.#Natl.#Acad.#Sci.#USA#108,- Suppl.#1,-4631$4638.-

Padmanabhan,-M.,-Cournoyer,-P.,-and-Dinesh$Kumar,-S.P.-(2009).-The-Leucine$Rich-Repeat-

Domain-in-Plant-Innate-Immunity:-A-Wealth-of-Possibilities.-Cell#Microbiol.#11,-191$198.-

-

Appendix,A:,Site\Directed,Mutagenesis,Primer,Sequences,and,Locations, ,

,

Primer#List#

,

Primer,Name, Sequence, Description,

W500- TAC-GGC-ATC-CGT-TCG-CCA-CCG-TCG-C- 5’-IC2-Flank- W501- TGG-CAA-GCT-ATT-GGC-ATA-CGC-ATC- 3’-IC2-Flank- W502- GAG-CCA-GTG-TAC-GAC-AAC-TTG-GGA-TTG-C- 5’-IC3-Flank- W503- CAC-ACC-CGT-GGC-CGG-CGA-ATA-GAC- 3’-IC3-Flank- ,

Primer#Position#on#Kek5

, ,

, ,

Appendix,B:,Ongoing,Transgenic,F1,Crosses,in,Drosophila, ,

,

Kek5

,Transgenic,F1,Crosses, Kek5

,Transgenic,F1,Crosses,

1A$2F$1M- 2A$11F$1F-

1A$2F$2F- 2A$11F$2M-

1A$4F$1M- 2A$16F$1F-

1A$4F$2M- 2A$16F$2M-

1A$4F$3M- 2A$16F$3M-

1A$8M$1F- 2A$27F$1M-

1A$8M$2M- 2A$27F$2F-

1A$8M$3M- 2A$34F$1M-

1A$9F$1F- 2A$34F$2F-

1A$18M$1M- 2A$4F$1M-

1A$18M$2M- 2A$18F$1F-

1B$2F$1M- 2A$18F$2M-

1B$2F$2M- 2A$22M$1M-

1B$12M$1M- 2A$22M$2M-

1B$12M$2F- 2B$4F$1M-

- 2B$4F$2M-

- 2B$6M$1F-

- 2B$6M$2F-

- 2C$25M$1M-

, 2C$25M$2M-

, 2C$29F$1M-

,

Notes:%All-lines-were-crossed-with-w^1118-flies-of-the-opposite-sex.-The-first-section-of-each-transgenic-label- corresponds-to-the-vial-each-G0-originated-from.-The-second-section-of-each-label-corresponds-to-the-sex-of-the- parent-G0-and-the-order-in-which-it-eclosed-compared-to-its-siblings.-The-third-section-of-each-label-

corresponds-to-the-sex-of-each-F1-transgenic-and-the-order-in-which-it-eclosed.--

,,,,

, ,

Appendix,C:,GENE,CONSTRUCTION,CONSTRUCTS,

,

CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG

CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT

TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCAAAAAGGCCA TCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTT GCTTCACAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACG

AAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCGTTAACGCTAGCATG

GATCTCGGGCCCC

AAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAAT

TGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCT

ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG GCC CTA CCG CCG CCC ACC GCA GGC

ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC CAC TGT CAG TGG AAT TCG GGC AAG AAG CTCGGAAAGCAAAATAAACTACGGACCGTCAAGGGATGAGAGCGCAATTGCGATCGTAC

CTAGAGCCCGGGG

TTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTTTA

ACTACTCGTTACGAAAAAATATTACGGTTGAAACATGTTTTTTCGTCCGA

TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC CGG GAT GGC GGC GGG TGG CGT CCG

TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG GTG ACA GTC ACC TTA AGC CCG TTC TTC GCGGACCATAGAAATATCAGGACAGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCC

GCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGAGTGTACAAGA

AAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGATCGGTCCTTCTCAAACATCTTTGCGTTTTTCCGGT AGGCAGTCCTACCGGAAGACGAATCAAACTACGGACCGTCAAATACCGCCCGCAGGACGGGCGGTGGGAGGCCCGGCAA CGAAGTGTTGCAAGTTTAGGCGAGGGCCGCCTAAACAGGATGAGTCCTCTCGCAAGTGGCTGTTTGTTGTCTATTTTGC

TTTCCGGGTCAGAAGGCTGA

M

I L L L L G V L V V L M A L P P P T A G

T T D W M Q S C G T C H C Q W N S G K K

S A D C K N K A L T K I P Q D M S N E M

Q V L D F A H N Q I P E L R R E E F L L

A G L P N V H K I F L R N C T I Q E V H

R E A F K G L H I L I E L D L S G N R I

R E L H P G T F A G L E K L R N V I I N

N N E I E V L P N H L F V N L S F L S R

I E F R N N R L R Q V Q L H V F A G T M

A L S A I S L E Q N R L S H L H K E T F

K D L Q K L M H L S L Q G N A W N C S C

E L Q D F R D F A I S K R L Y T P P T D

C Q E P P Q L R G K L W S E V P S E N F

A C R P R I L G S V R S F I E A N H D N

I S L P C R I V G S P R P N V T W V Y N

K R P L Q Q Y D P R V R V L T S V E Q M

P E Q P S Q V L T S E L R I V G V R A S

D K G A Y T C V A D N R G G R A E A E F

Q L L V S G D Y A G A V S A S D G M G M

G A I G A P T I D P Q T N M F L I I C L

I I T T L L L L L L V A V L T L F W Y C

R R I K T Y Q K D T T M M S G D G L I S

S K M D K T H N G S M L E G S V I M E M

Q K S L L N E V N P V E K P P R R T D I

E S V D G G D D V L E I K K T L L D D T

V Y V A N H S R D E E A V S V A M S D T

T T T P R S R H T Y V D D A Y A N S L P

Y G I R S P P S L T S P V Y T H M T P H

G I Y G T K T M T A P H N G F M T L Q H

P K S R N L A L I A T T N S S R Q H Q H

H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q

Q Q Q Q Q H P L A T T S P F L P A P V V

Y S P A T G V V M K Q G Y M T I P R K P

R A P S W A P S T S G A A G H G S I Q L

S E F Q S P T S P N P S E T G T A T T A

E L Q A E P V Y D N L G L R T T A G G N

S T L N L T K I A G S Q G G A G Q Q Y S

M R D R P L P A T P S L T S V S S A T N

A S K I Y E P I H E L I Q Q Q Q Q L Q Q

Q Q Q Q Q Q Q R L G S M D T E P L Y G V

R Q Q G I T I L P G S S I S G A G L G H

A A Y L S P G S G A A V S P S H A S S S

G D S P K A A K I P P R P P P K P K K K

M S V T T T R S G Q G S T S Q L F D D E

W50

Start of Good Sequence (B1, pENTRK5∆2)

NheI

NheI

attL1

junction marker

signal sequence

KEK5

N FLANK 1

8 0

159 238 317

396

475

537

598

658

1

2

2 2

4 2

6 2

8 2

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862

AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG

CAG GTG CTG GAC TTT GCC CAC AAT CAA ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG

GCC GGT CTG CCC AAT GTG CAC AAG ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT

CGC GAG GCC TTC AAG GGT CTG CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA

CGG GAA CTG CAT CCG GGC ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gT G ATC ATC AAC

AAC AAC GAG ATC GAG GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC

ATC GAG TTC CGG AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG

GCG CTG AGC GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC

GTC CAC GAC CTG AAA CGG GTG TTA GTT TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC

CGG CCA GAC GGG TTA CAC GTG TTC TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA

GCG CTC CGG AAG TTC CCA GAC GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT

GCC CTT GAC GTA GGC CCG TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cA C TAG TAG TTG

TTG TTG CTC TAG CTC CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG

TAG CTC AAG GCC TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC

CGC GAC TCG CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG M

I L L L L G V L V V L M A L P P P T A G

T T D W M Q S C G T C H C Q W N S G K K

S A D C K N K A L T K I P Q D M S N E M

Q V L D F A H N Q I P E L R R E E F L L

A G L P N V H K I F L R N C T I Q E V H

R E A F K G L H I L I E L D L S G N R I

R E L H P G T F A G L E K L R N V I I N

N N E I E V L P N H L F V N L S F L S R

I E F R N N R L R Q V Q L H V F A G T M

A L S A I S L E Q N R L S H L H K E T F

K D L Q K L M H L S L Q G N A W N C S C

E L Q D F R D F A I S K R L Y T P P T D

C Q E P P Q L R G K L W S E V P S E N F

A C R P R I L G S V R S F I E A N H D N

I S L P C R I V G S P R P N V T W V Y N

K R P L Q Q Y D P R V R V L T S V E Q M

P E Q P S Q V L T S E L R I V G V R A S

D K G A Y T C V A D N R G G R A E A E F

Q L L V S G D Y A G A V S A S D G M G M

G A I G A P T I D P Q T N M F L I I C L

I I T T L L L L L L V A V L T L F W Y C

R R I K T Y Q K D T T M M S G D G L I S

S K M D K T H N G S M L E G S V I M E M

Q K S L L N E V N P V E K P P R R T D I

E S V D G G D D V L E I K K T L L D D T

V Y V A N H S R D E E A V S V A M S D T

T T T P R S R H T Y V D D A Y A N S L P

Y G I R S P P S L T S P V Y T H M T P H

G I Y G T K T M T A P H N G F M T L Q H

P K S R N L A L I A T T N S S R Q H Q H

H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q

Q Q Q Q Q H P L A T T S P F L P A P V V

Y S P A T G V V M K Q G Y M T I P R K P

R A P S W A P S T S G A A G H G S I Q L

S E F Q S P T S P N P S E T G T A T T A

E L Q A E P V Y D N L G L R T T A G G N

S T L N L T K I A G S Q G G A G Q Q Y S

M R D R P L P A T P S L T S V S S A T N

A S K I Y E P I H E L I Q Q Q Q Q L Q Q

Q Q Q Q Q Q Q R L G S M D T E P L Y G V

R Q Q G I T I L P G S S I S G A G L G H

A A Y L S P G S G A A V S P S H A S S S

G D S P K A A K I P P R P P P K P K K K

M S V T T T R S G Q G S T S Q L F D D E LRRs

BglII

Actual sequence is GTG - should match original wt sequence

oligo238 718

778

838

898

958

1018

1078

1138

1

2

2 2

4 2

6 2

8 2

102

122

142

162

182

202

222

242

262

282

302

322

342

362

382

402

422

442

462

482

502

522

542

562

582

602

622

642

662

682

702

722

742

762

782

802

822

842

862