Christian Francom, Jeffrey Caldwell, Barbara Trask, Ph.D.

Extracellular Matrix Functions Structure—tense or elastic Cellular Adhesion Cellular Motility Molecular Reservoir

Elastic Extracellular Matrices Component parts Elastin Microfibrils Fibrillins MAGPs Fibulins Other proteins and proteoglycans Disorders involving the Elastic ECM Elastin SVAS Microfibrils Marfan Syndrome

Question How 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 Organism Zebrafish (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

Stages of Z-fish Development

Sequences and Primers Fibrillin atggcagcctgtcctgaattaatcttggaaagagagggaggagaaggagtggatgggggattctttaaggcgaagatgactaaggat ttggtggaacgggagattcgcatcacagatgtgcagccggcaaatctgcagcaactgcgtgatgctgtcatctcaaaattggccaaaa tctctgaggaatatttccagtaccttgttgaatctttgccacaaagaattgaggcatttgtgaaagcaaaaaggatgtccaacctggag ggagccccaggctccaggatattctcagctcaggatgtgaacgagtgcaacgaccccaacacctgcatcagcggtctgtgtctgaac acccctggcagctacatctgcagctgcccccccgactttgagctcaatcccactggagtcggctgcgtcgacactcgctctggaaact gctacctggacgtccagtaccgaggcgatttctctgagagtctgagctgctccaatgagattggagtgggagtgtccaaagcgtcctg ctgctgttctaggggtgtggcctggggacagccctgcgaggcctgtccatcactcaactccaagatctgtttcactgcacaacctgaaa tctccagtctggtgcagggagagaccagccagtgcttccccaggaggagtatccagtgctgggacacattagcggctcattcccttgg gcaagcctggagctcagatggccagctgcacgtttccactcttcatctttgttgtggtgtgaaagaggagaaggagggtttaggaaaa caaaaaagcagtggacagagtgatgttacaaagacaacaggcttcacacagagctctgacagtcgcgtcaaagcgagtgcgctgtta ttaaaatggtaccgtcccagtgacagctctcacagctttatttttgcagtgtcaggactacggtccaatcatgcaatagtagaggacatc cgtctgttcaacagtaagcaatatggagctgagccgctgaaagaactgctcaaactactaccagaagcagatgaggtgaagagattg agagaattcaaaggagacgcaaataagctgacactggttgattccttcatgttccagctcattcaagtaccacgctttgatgtgcgtatc gaggtcatggtacttcaagaagagtttttcccattatgtactgccatgagtcgtgacatcaacattgtgcgccaggcaactgaagctgt gcctgtattagccttctctaagaaaccaatggctcatgaccttttagtcctgacaggtggctatgcaggaaacgctgtaggcttcaaatt gtcctctcttctctctctggctgacacaaaggccaacaagcctggcatgaacctgctccattttgtggcattactctcgaggatcttctct gtgttttcacagggtggctatgcaggaaacgctgtaggcttcaaattgtcctctcttctctctctggctgacacaaaggccaacaagcct ggcatgaacctgctccattttgtggcattagaagcacagaaaaaggatgaggcacttttaaagttcccagagaagctaacacatattc agagtgcagccagaatttcagtagaaaatatagaaggagagttttcctcactctatgtgaaaacacggtctttggaacagaagattcaa gatgacgatgagctgcaaaagcagctagatccatttttgcagagctctgcaaaagccttgcaggaccttaagcaccgcaggctggact tgcgcaaagagggtaatgccctcattgacttcttctgtgaagacaaagacaccttcaaattggatgagtgcttcagaatcttccaagatt tctgtctaaagttcaaaaaagctgtgaaggacaactcggacagagaactgaaagaggaagctagacagcgccgcttacgggaacta gaggagagacggtgtgcctggggtgcagccaatattgaaacaggtggtaatggaggttttggtcgcagcagcagtgaaaatgatgtt gacatcttaaccaaagatggcctcctagacttcctgcttcaatcaagacctcaaagcccccacagtcctttaggacgctcagctagtgc tcgccgctaccaccacacatctagtgaccgcagcctccgtggctatatggagctggtctctgggtcttcagcaagtgaatacgccaaa ttcagcagcctgccacgttcaagcaggaaccatcaaagaaagaccatgccatggcttgtcttgcaggatgataatagagagttgattc cacaaagtcaggtttaccaagagaagttagcagtttcacaaagggttgagactgaaccaatcagtcctttggcaagatactcaaattta ggctacaacttgaacagtgatccctatagtaataataataacaattatagttctctttctgaaggtggtcttacacctcgattaaataaaa caaagaatgtctttcaggggacatctggtcaaggcattggacacatgaatgtttctgttgagaaacgcaaactagtgcctgggcttcaa ccctttgagataaccagccagatcaataataacagtcatgtttctttggacaatcaacatgaagtgtgtgtgactgatctggaacgaga agaccttcccaaagcttttgttcttgatacaccaccatctagtagaagttctgaggaggggcataagctagaaaaagtatggaatgaca aaatcatctcatcacagagagacgaagcagacacaagtaccttctcttcaacatcttgtgacaccccattgccccttgatccctccagt aaaaaacctgtgttttatgtcatggactgcactgaaacggattgttccattgtgttggactgttctgagattgagagttctcctattataa aagaaggacctggacttatcccaaagttacaggatgtcaactttccaagaatcattcaagacaccaattctctttcatccaattttgagt cttcagactgtcactttgtgtccacatcaagtgatgaaccatttgtaggaagggtcgatcaaacttcagaatcagtcagtccatctgtaa caactgaagaagcagagacagacagctgtgacacagctgaagggcaaagagttgatgagaaaggtgttcagacaggtagccccaaa acgacacttatcaaaaccaagacttcatccaaagggatgactgctcacaccagccgtcctacaagaaccctcacagctactgagactc agaacatgcgaaaagtagtgcccattacaaggcaaaatcgatctagtagcagcatgagaaaggttgataaaccacctgtacatgaga gctctgaacccagacgtccatttcgagaccaaagcatgccagccagaagaggcgagagacaaggtagacctacaagacattcaagc cttccaccagaggatcctaggacaccaagaggttcgacaccaagcagctctcgttggggaagggactcaattcaacacaggccctcc atcaagaaagccaatgccaaacctgttcgtaacattcctaaaccagcaccagaggaaaagatgtgtcgctctacaatgcgtgccttgg ctcaagctcaggctcaaggaacctcagaaggaggtaccgctgacagccctaacaccaccccaaaaagttcttcacctttgcctagctt tgctcgcaacactgtggcatctacttctagatctaaaaaagacatgccatcagcaactgtaaccggaacaccatcaaagagcctatcg agaactagctctcagagattgcctggtggtagaacagagtctgtcagtagcgggcctttaccaaggagtgaggataaactaggaggct cgattaggagggttcaaagtgtaagagcttccagtcgaagcagtgacacaccttcacaatctggtcgggatcactcccttaaaagtag cagtttctcagacagatccacaccaactagagacaccatcatctccagcaggaccagcaaacccacatggaaatag MAGP atgagactctgttacctgcttctgctgtcactgccgggtctgctggctcaggtgcagaactat gatgaggattatacagacatcttcggtaacagcaacaactacgacaaccccttcttctacca gcagcagatattacagacgcagacgtcagggcctgaacagaacgcgctggatggacagac agagccgactgaacctggaccgctggactgtcgtgaggagcagtatccctgcactcggctc tattctgtccatcagccgtgtaaacagtgtctgaacagcatctgcttctacagtctgcgcagg atgtatgtgatcaataaggaggtgtgtgtgcggacggtgtgtgcacatgaggagctcttgag agcggatctgtgtcgtgaccagttcccccgctgtggtgtaatggcgcacattggccagtgtg gggcgatggggagcaactgcgccaggagttgtggatcctgctaa

Expression Data Fibrillin MAGP GAPDH Control

Future Possibilities More replicates should be done Analysis with Real-Time PCR Alternative primers should be made In situ hybridization should localize expression

Question How 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 interactions Fibrillin interacts with: MAGP interacts with:  MAGP  CSPG  LTGF-β  Others?  Fibrillins  CSPG  Elastin  Others?

Mammalian Structure of Fibrillin-1

Isolated Bands of LTGF-β Domains bp 200bp 100bp

LTGF-β Binding Domains ….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVE SLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVG C VDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQ PCEACPSLNSKICFTAQPEISSLVQGET SQCFPRRSIQCWDTLAAHSLGQAWSSDGQ LHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGL RSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEV MVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKA NKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALL KFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKAL QDLKHRRL DLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEE ARQR RLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASA RRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLA VSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSV EKRKL VPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPS SRSSEEGH KLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCS EIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEA ETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSS SSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHR PSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTS RSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSG RDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK

LTGF-β Binding Domains ….MAACPELILEREGGEGVDGGFFKAKMTKDLVEREIRITDVQPANLQQLRDAVISKLAKISEEYFQYLVE SLPQRIEAFVKAKRMSNLEGAPGSRIFSAQDVNECNDPNTCISGLCLNTPGSYICSCPPDFELNPTGVG C VDTRSGNCYLDVQYRGDFSESLSCSNEIGVGVSKASCCCSRGVAWGQ PCEACPSLNSKICFTAQPEISSLVQGET SQCFPRRSIQCWDTLAAHSLGQAWSSDGQ LHVSTLHLCCGVKEEKEGLGKQKSSGQSDVTKTTGFTQSSDSRVKASALLLKWYRPSDSSHSFIFAVSGL RSNHAIVEDIRLFNSKQYGAEPLKELLKLLPEADEVKRLREFKGDANKLTLVDSFMFQLIQVPRFDVRIEV MVLQEEFFPLCTAMSRDINIVRQATEAVPVLAFSKKPMAHDLLVLTGGYAGNAVGFKLSSLLSLADTKA NKPGMNLLHFVALLSRIFSVFSQGGYAGNAVGFKLSSLLSLADTKANKPGMNLLHFVALEAQKKDEALL KFPEKLTHIQSAARISVENIEGEFSSLYVKTRSLEQKIQDDDELQKQLDPFLQSSAKAL QDLKHRRL DLRKEGNALIDFFCEDKDTFKLDECFRIFQDFCLKFKKAVKDNSDRELKEE ARQR RLRELEERRCAWGAANIETGGNGGFGRSSSENDVDILTKDGLLDFLLQSRPQSPHSPLGRSASA RRYHHTSSDRSLRGYMELVSGSSASEYAKFSSLPRSSRNHQRKTMPWLVLQDDNRELIPQSQVYQEKLA VSQRVETEPISPLARYSNLGYNLNSDPYSNNNNNYSSLSEGGLTPRLNKTKNVFQGTSGQGIGHMNVSV EKRKL VPGLQPFEITSQINNNSHVSLDNQHEVCVTDLEREDLPKAFVLDTPPS SRSSEEGH KLEKVWNDKIISSQRDEADTSTFSSTSCDTPLPLDPSSKKPVFYVMDCTETDCSIVLDCS EIESSPIIKEGPGLIPKLQDVNFPRIIQDTNSLSSNFESSDCHFVSTSSDEPFVGRVDQTSESVSPSVTTEEA ETDSCDTAEGQRVDEKGVQTGSPKTTLIKTKTSSKGMTAHTSRPTRTLTATETQNMRKVVPITRQNRSS SSMRKVDKPPVHESSEPRRPFRDQSMPARRGERQGRPTRHSSLPPEDPRTPRGSTPSSSRWGRDSIQHR PSIKKANAKPVRNIPKPAPEEKMCRSTMRALAQAQAQGTSEGGTADSPNTTPKSSSPLPSFARNTVASTS RSKKDMPSATVTGTPSKSLSRTSSQRLPGGRTESVSSGPLPRSEDKLGGSIRRVQSVRASSRSSDTPSQSG RDHSLKSSSFSDRSTPTRDTIISSRTSKPTWK

Future Directions Clone for expression Protein binding studies

Acknowledgements Josh McBride Aric Elmer OUR