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Structure, function, and temporal expression of Fibrillin in Danio rerio. Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D. Extracellular Matrix. Functions Structure—tense or elastic Cellular Adhesion Cellular Motility Molecular Reservoir. Elastic Extracellular Matrices. - PowerPoint PPT Presentation
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Structure, function, and temporal expression of Fibrillin in Danio rerio.
Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D.
Extracellular MatrixFunctions
Structure—tense or elasticCellular AdhesionCellular MotilityMolecular Reservoir
Elastic Extracellular MatricesComponent parts
ElastinMicrofibrils
Fibrillins MAGPs Fibulins Other proteins and proteoglycans
Disorders involving the Elastic ECMElastin SVASMicrofibrils Marfan Syndrome
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?
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 maintainedhttp://www.pdn.cam.ac.uk/staff/lewis/zfish3AgesSlide.jpg
Stages of Z-fish Development
Sequences and PrimersFibrillin
atggcagcctgtcctgaattaatcttggaaagagagggaggagaaggagtggatgggggattctttaaggcgaagatgactaaggatttggtggaacgggagattcgcatcacagatgtgcagccggcaaatctgcagcaactgcgtgatgctgtcatctcaaaattggccaaaatctctgaggaatatttccagtaccttgttgaatctttgccacaaagaattgaggcatttgtgaaagcaaaaaggatgtccaacctggagggagccccaggctccaggatattctcagctcaggatgtgaacgagtgcaacgaccccaacacctgcatcagcggtctgtgtctgaacacccctggcagctacatctgcagctgcccccccgactttgagctcaatcccactggagtcggctgcgtcgacactcgctctggaaactgctacctggacgtccagtaccgaggcgatttctctgagagtctgagctgctccaatgagattggagtgggagtgtccaaagcgtcctgctgctgttctaggggtgtggcctggggacagccctgcgaggcctgtccatcactcaactccaagatctgtttcactgcacaacctgaaatctccagtctggtgcagggagagaccagccagtgcttccccaggaggagtatccagtgctgggacacattagcggctcattcccttgggcaagcctggagctcagatggccagctgcacgtttccactcttcatctttgttgtggtgtgaaagaggagaaggagggtttaggaaaacaaaaaagcagtggacagagtgatgttacaaagacaacaggcttcacacagagctctgacagtcgcgtcaaagcgagtgcgctgttattaaaatggtaccgtcccagtgacagctctcacagctttatttttgcagtgtcaggactacggtccaatcatgcaatagtagaggacatccgtctgttcaacagtaagcaatatggagctgagccgctgaaagaactgctcaaactactaccagaagcagatgaggtgaagagattgagagaattcaaaggagacgcaaataagctgacactggttgattccttcatgttccagctcattcaagtaccacgctttgatgtgcgtatcgaggtcatggtacttcaagaagagtttttcccattatgtactgccatgagtcgtgacatcaacattgtgcgccaggcaactgaagctgtgcctgtattagccttctctaagaaaccaatggctcatgaccttttagtcctgacaggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattactctcgaggatcttctctgtgttttcacagggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattagaagcacagaaaaaggatgaggcacttttaaagttcccagagaagctaacacatattcagagtgcagccagaatttcagtagaaaatatagaaggagagttttcctcactctatgtgaaaacacggtctttggaacagaagattcaagatgacgatgagctgcaaaagcagctagatccatttttgcagagctctgcaaaagccttgcaggaccttaagcaccgcaggctggacttgcgcaaagagggtaatgccctcattgacttcttctgtgaagacaaagacaccttcaaattggatgagtgcttcagaatcttccaagatttctgtctaaagttcaaaaaagctgtgaaggacaactcggacagagaactgaaagaggaagctagacagcgccgcttacgggaactagaggagagacggtgtgcctggggtgcagccaatattgaaacaggtggtaatggaggttttggtcgcagcagcagtgaaaatgatgttgacatcttaaccaaagatggcctcctagacttcctgcttcaatcaagacctcaaagcccccacagtcctttaggacgctcagctagtgctcgccgctaccaccacacatctagtgaccgcagcctccgtggctatatggagctggtctctgggtcttcagcaagtgaatacgccaaattcagcagcctgccacgttcaagcaggaaccatcaaagaaagaccatgccatggcttgtcttgcaggatgataatagagagttgattccacaaagtcaggtttaccaagagaagttagcagtttcacaaagggttgagactgaaccaatcagtcctttggcaagatactcaaatttaggctacaacttgaacagtgatccctatagtaataataataacaattatagttctctttctgaaggtggtcttacacctcgattaaataaaacaaagaatgtctttcaggggacatctggtcaaggcattggacacatgaatgtttctgttgagaaacgcaaactagtgcctgggcttcaaccctttgagataaccagccagatcaataataacagtcatgtttctttggacaatcaacatgaagtgtgtgtgactgatctggaacgagaagaccttcccaaagcttttgttcttgatacaccaccatctagtagaagttctgaggaggggcataagctagaaaaagtatggaatgacaaaatcatctcatcacagagagacgaagcagacacaagtaccttctcttcaacatcttgtgacaccccattgccccttgatccctccagtaaaaaacctgtgttttatgtcatggactgcactgaaacggattgttccattgtgttggactgttctgagattgagagttctcctattataaaagaaggacctggacttatcccaaagttacaggatgtcaactttccaagaatcattcaagacaccaattctctttcatccaattttgagtcttcagactgtcactttgtgtccacatcaagtgatgaaccatttgtaggaagggtcgatcaaacttcagaatcagtcagtccatctgtaacaactgaagaagcagagacagacagctgtgacacagctgaagggcaaagagttgatgagaaaggtgttcagacaggtagccccaaaacgacacttatcaaaaccaagacttcatccaaagggatgactgctcacaccagccgtcctacaagaaccctcacagctactgagactcagaacatgcgaaaagtagtgcccattacaaggcaaaatcgatctagtagcagcatgagaaaggttgataaaccacctgtacatgagagctctgaacccagacgtccatttcgagaccaaagcatgccagccagaagaggcgagagacaaggtagacctacaagacattcaagccttccaccagaggatcctaggacaccaagaggttcgacaccaagcagctctcgttggggaagggactcaattcaacacaggccctccatcaagaaagccaatgccaaacctgttcgtaacattcctaaaccagcaccagaggaaaagatgtgtcgctctacaatgcgtgccttggctcaagctcaggctcaaggaacctcagaaggaggtaccgctgacagccctaacaccaccccaaaaagttcttcacctttgcctagctttgctcgcaacactgtggcatctacttctagatctaaaaaagacatgccatcagcaactgtaaccggaacaccatcaaagagcctatcgagaactagctctcagagattgcctggtggtagaacagagtctgtcagtagcgggcctttaccaaggagtgaggataaactaggaggctcgattaggagggttcaaagtgtaagagcttccagtcgaagcagtgacacaccttcacaatctggtcgggatcactcccttaaaagtagcagtttctcagacagatccacaccaactagagacaccatcatctccagcaggaccagcaaacccacatggaaatag
MAGPatgagactctgttacctgcttctgctgtcactgccgggtctgctggctcaggtgcagaactatgatgaggattatacagacatcttcggtaacagcaacaactacgacaaccccttcttctaccagcagcagatattacagacgcagacgtcagggcctgaacagaacgcgctggatggacagacagagccgactgaacctggaccgctggactgtcgtgaggagcagtatccctgcactcggctctattctgtccatcagccgtgtaaacagtgtctgaacagcatctgcttctacagtctgcgcaggatgtatgtgatcaataaggaggtgtgtgtgcggacggtgtgtgcacatgaggagctcttgagagcggatctgtgtcgtgaccagttcccccgctgtggtgtaatggcgcacattggccagtgtggggcgatggggagcaactgcgccaggagttgtggatcctgctaa
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
Rela
tive
Inte
nsity
Fibrillin
0
50
100
150
200
8 Som 21Som
24 hpf 35 hpf 52 hpf 75 hpf 96 hpf 120 hpf
Developmental Timepoint
Aver
age
Rela
tive
Inte
nsity
MAGPGAPDH Control
Future PossibilitiesMore replicates should be done
Analysis with Real-Time PCRAlternative primers should be madeIn situ hybridization should localize
expression
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?
Intermolecular interactionsFibrillin interacts with: MAGP interacts with:
MAGP CSPG LTGF-β Others?
Fibrillins CSPG Elastin Others?
Mammalian Structure of Fibrillin-1
Fibrillin-1
3 5 6
Isolated Bands of LTGF-β Domains
3 5 6
300bp200bp100bp
LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVESLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK
LTGF-β Binding Domains….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVESLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVGCVDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQPCEACPSLNSKICFTAQPEISSLVQGETSQCFPRRSIQCWDTLAAHSLGQAWSSDGQLHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGLRSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEVMVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALLKFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKALQDLKHRRLDLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEEARQRRLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASARRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLAVSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSVEKRKLVPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPSSRSSEEGHKLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCSEIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEAETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSSSSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHRPSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTSRSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSGRDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK
Future DirectionsClone for expressionProtein binding studies
AcknowledgementsJosh McBrideAric ElmerOUR