christian francom, jeffrey caldwell, barbara trask, ph.d
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![Page 1: Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D](https://reader035.vdocuments.us/reader035/viewer/2022062421/56649d2c5503460f94a02237/html5/thumbnails/1.jpg)
Structure, function, and temporal expression of Fibrillin in Danio
rerio.
Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D.
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Extracellular MatrixFunctions
Structure—tense or elasticCellular AdhesionCellular MotilityMolecular Reservoir
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Elastic Extracellular MatricesComponent parts
ElastinMicrofibrils
Fibrillins MAGPs Fibulins Other proteins and proteoglycans
Disorders involving the Elastic ECMElastin SVASMicrofibrils Marfan Syndrome
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QuestionHow do the microfibrillar proteins contribute
to the function of the elastic extracellular matrix?When and where is each expressed?What inter-molecular interactions are
maintained?
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Model OrganismZebrafish (Danio rerio)
External fertilization and development
Colorless during the first few days of development
Genome sequenced (although not assembled)
**first evolutionary organism to have functional elastic extracellular matrix
Easily maintained
http://www.pdn.cam.ac.uk/staff/lewis/zfish3AgesSlide.jpg
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Stages of Z-fish Development
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Sequences and PrimersFibrillin
atggcagcctgtcctgaattaatcttggaaagagagggaggagaaggagtggatgggggattctttaaggcgaagatgactaaggatttggtggaacgggagattcgcatcacagatgtgcagccggcaaatctgcagcaactgcgtgatgctgtcatctcaaaattggccaaaatctctgaggaatatttccagtaccttgttgaatctttgccacaaagaattgaggcatttgtgaaagcaaaaaggatgtccaacctggagggagccccaggctccaggatattctcagctcaggatgtgaacgagtgcaacgaccccaacacctgcatcagcggtctgtgtctgaacacccctggcagctacatctgcagctgcccccccgactttgagctcaatcccactggagtcggctgcgtcgacactcgctctggaaactgctacctggacgtccagtaccgaggcgatttctctgagagtctgagctgctccaatgagattggagtgggagtgtccaaagcgtcctgctgctgttctaggggtgtggcctggggacagccctgcgaggcctgtccatcactcaactccaagatctgtttcactgcacaacctgaaatctccagtctggtgcagggagagaccagccagtgcttccccaggaggagtatccagtgctgggacacattagcggctcattcccttgggcaagcctggagctcagatggccagctgcacgtttccactcttcatctttgttgtggtgtgaaagaggagaaggagggtttaggaaaacaaaaaagcagtggacagagtgatgttacaaagacaacaggcttcacacagagctctgacagtcgcgtcaaagcgagtgcgctgttattaaaatggtaccgtcccagtgacagctctcacagctttatttttgcagtgtcaggactacggtccaatcatgcaatagtagaggacatccgtctgttcaacagtaagcaatatggagctgagccgctgaaagaactgctcaaactactaccagaagcagatgaggtgaagagattgagagaattcaaaggagacgcaaataagctgacactggttgattccttcatgttccagctcattcaagtaccacgctttgatgtgcgtatcgaggtcatggtacttcaagaagagtttttcccattatgtactgccatgagtcgtgacatcaacattgtgcgccaggcaactgaagctgtgcctgtattagccttctctaagaaaccaatggctcatgaccttttagtcctgacaggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattactctcgaggatcttctctgtgttttcacagggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattagaagcacagaaaaaggatgaggcacttttaaagttcccagagaagctaacacatattcagagtgcagccagaatttcagtagaaaatatagaaggagagttttcctcactctatgtgaaaacacggtctttggaacagaagattcaagatgacgatgagctgcaaaagcagctagatccatttttgcagagctctgcaaaagccttgcaggaccttaagcaccgcaggctggacttgcgcaaagagggtaatgccctcattgacttcttctgtgaagacaaagacaccttcaaattggatgagtgcttcagaatcttccaagatttctgtctaaagttcaaaaaagctgtgaaggacaactcggacagagaactgaaagaggaagctagacagcgccgcttacgggaactagaggagagacggtgtgcctggggtgcagccaatattgaaacaggtggtaatggaggttttggtcgcagcagcagtgaaaatgatgttgacatcttaaccaaagatggcctcctagacttcctgcttcaatcaagacctcaaagcccccacagtcctttaggacgctcagctagtgctcgccgctaccaccacacatctagtgaccgcagcctccgtggctatatggagctggtctctgggtcttcagcaagtgaatacgccaaattcagcagcctgccacgttcaagcaggaaccatcaaagaaagaccatgccatggcttgtcttgcaggatgataatagagagttgattccacaaagtcaggtttaccaagagaagttagcagtttcacaaagggttgagactgaaccaatcagtcctttggcaagatactcaaatttaggctacaacttgaacagtgatccctatagtaataataataacaattatagttctctttctgaaggtggtcttacacctcgattaaataaaacaaagaatgtctttcaggggacatctggtcaaggcattggacacatgaatgtttctgttgagaaacgcaaactagtgcctgggcttcaaccctttgagataaccagccagatcaataataacagtcatgtttctttggacaatcaacatgaagtgtgtgtgactgatctggaacgagaagaccttcccaaagcttttgttcttgatacaccaccatctagtagaagttctgaggaggggcataagctagaaaaagtatggaatgacaaaatcatctcatcacagagagacgaagcagacacaagtaccttctcttcaacatcttgtgacaccccattgccccttgatccctccagtaaaaaacctgtgttttatgtcatggactgcactgaaacggattgttccattgtgttggactgttctgagattgagagttctcctattataaaagaaggacctggacttatcccaaagttacaggatgtcaactttccaagaatcattcaagacaccaattctctttcatccaattttgagtcttcagactgtcactttgtgtccacatcaagtgatgaaccatttgtaggaagggtcgatcaaacttcagaatcagtcagtccatctgtaacaactgaagaagcagagacagacagctgtgacacagctgaagggcaaagagttgatgagaaaggtgttcagacaggtagccccaaaacgacacttatcaaaaccaagacttcatccaaagggatgactgctcacaccagccgtcctacaagaaccctcacagctactgagactcagaacatgcgaaaagtagtgcccattacaaggcaaaatcgatctagtagcagcatgagaaaggttgataaaccacctgtacatgagagctctgaacccagacgtccatttcgagaccaaagcatgccagccagaagaggcgagagacaaggtagacctacaagacattcaagccttccaccagaggatcctaggacaccaagaggttcgacaccaagcagctctcgttggggaagggactcaattcaacacaggccctccatcaagaaagccaatgccaaacctgttcgtaacattcctaaaccagcaccagaggaaaagatgtgtcgctctacaatgcgtgccttggctcaagctcaggctcaaggaacctcagaaggaggtaccgctgacagccctaacaccaccccaaaaagttcttcacctttgcctagctttgctcgcaacactgtggcatctacttctagatctaaaaaagacatgccatcagcaactgtaaccggaacaccatcaaagagcctatcgagaactagctctcagagattgcctggtggtagaacagagtctgtcagtagcgggcctttaccaaggagtgaggataaactaggaggctcgattaggagggttcaaagtgtaagagcttccagtcgaagcagtgacacaccttcacaatctggtcgggatcactcccttaaaagtagcagtttctcagacagatccacaccaactagagacaccatcatctccagcaggaccagcaaacccacatggaaatag
MAGPatgagactctgttacctgcttctgctgtcactgccgggtctgctggctcaggtgcagaactatgatgaggattatacagacatcttcggtaacagcaacaactacgacaaccccttcttctaccagcagcagatattacagacgcagacgtcagggcctgaacagaacgcgctggatggacagacagagccgactgaacctggaccgctggactgtcgtgaggagcagtatccctgcactcggctctattctgtccatcagccgtgtaaacagtgtctgaacagcatctgcttctacagtctgcgcaggatgtatgtgatcaataaggaggtgtgtgtgcggacggtgtgtgcacatgaggagctcttgagagcggatctgtgtcgtgaccagttcccccgctgtggtgtaatggcgcacattggccagtgtggggcgatggggagcaactgcgccaggagttgtggatcctgctaa
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Expression DataFibrillin MAGP
Fibrillin
Actin Control
MAGP
0
50
100
150
200
8 Som 21 Som 24 hpf 35 hpf 52 hpf 75 hpf 96 hpf 120 hpf
Developmental Timepoint
Rel
ativ
e In
ten
sity
Fibrillin
0
50
100
150
200
8 Som 21Som
24 hpf 35 hpf 52 hpf 75 hpf 96 hpf 120 hpf
Developmental Timepoint
Ave
rag
e R
elat
ive
Inte
nsi
ty
MAGP
GAPDH Control
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Future PossibilitiesMore replicates should be done
Analysis with Real-Time PCRAlternative primers should be madeIn situ hybridization should localize
expression
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QuestionHow do the microfibrillar proteins contribute
to the function of the elastic extracellular matrix?When and where is each expressed?What inter-molecular interactions are
maintained?
![Page 11: Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D](https://reader035.vdocuments.us/reader035/viewer/2022062421/56649d2c5503460f94a02237/html5/thumbnails/11.jpg)
Intermolecular interactionsFibrillin interacts with:
MAGP interacts with: MAGP CSPG LTGF-β Others?
Fibrillins CSPG Elastin Others?
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Mammalian Structure of Fibrillin-1
Fibrillin-1
3 5 6
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Isolated Bands of LTGF-β Domains
3 5 6
300bp
200bp
100bp
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LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVES
LPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEK
LTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETE
PISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK
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LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVES
LPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEK
LTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETE
PISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK
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Future DirectionsClone for expressionProtein binding studies
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AcknowledgementsJosh McBrideAric ElmerOUR