Monika Abedin

University of California, San Francisco

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Nicole King

Howard Hughes Medical Institute

Susan L. Young

United States Food and Drug Administration

Martin Carr

University of Huddersfield

B. S. C. Leadbeater

University of Birmingham

Asaf Salamov

Joint Genome Institute

Antonis Rokas

Vanderbilt University

Igor V. Grigoriev

University of California, Berkeley

Galo García

University of California, San Francisco

JGI Sequencing

Joint Genome Institute

M. Jody Westbrook

Alberta Children's Hospital

Cooperative Institutions

University of California, Berkeley

University of California, San Francisco

Joint Genome Institute

Howard Hughes Medical Institute

Lawrence Berkeley National Laboratory

Innovative Genomics Institute

United States Department of Energy

Cardiovascular Institute Hospital

Harvard University

Dartmouth College

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Preparation of High-Molecular-Weight Genomic DNA from Monosiga brevicollis and Other Choanoflagellates

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

genomic DNA

Isolation

10.1101/pdb.prot5153

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Citations (4)

Isolation of Single Choanoflagellate Cells from Field Samples and Establishment of Clonal Cultures

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

INTRODUCTION Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. This protocol describes how to enrich freshly collected field samples and isolate single choanoflagellate cells. Two methods are commonly used: isolation by micropipetting single cells and isolation by dilution. The two methods are complementary and each has its advantages and disadvantages. The micropipette technique requires considerable experience. However, provided a single cell has been isolated, any culture arising from it will be clonal. The dilution technique does not require the same level of skill, but single-cell isolation is not ensured. For the latter technique, repeating the isolation procedure using higher dilutions is advised to ensure that only one cell is the basis of an established culture.

Isolation

Serial dilution

Pipette

Dilution

10.1101/pdb.prot5147

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Citations (6)

Dynamic regulation of alternative ATP-binding cassette transporter A1 transcripts

Biochemical and Biophysical Research Communications (2003)

Jarkko Huuskonen Monika Abedin Meeta Vishnu Clive R. Pullinger Sergio E. Baranzini

Transcription

10.1016/s0006-291x(03)00992-6

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Citations (11)

Long-Term Frozen Storage of Choanoflagellate Cultures

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

Liquid nitrogen

10.1101/pdb.prot5149

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Citations (9)

Diverse evolutionary paths to cell adhesion

Trends in Cell Biology (2010)

Monika Abedin Nicole King

Multicellular organism

Lineage (genetic)

Slime Mold

10.1016/j.tcb.2010.08.002

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Citations (224)

The evolution of ciliary signaling mechanisms

Cilia (2012)

Monika Abedin JFR Reiter

Cilia, evolutionarily ancient structures that project from the cell surface into the extracellular environment, are critical sensors of external cues. In animals, they detect light, odors, soluble chemicals and mechanical forces, and defects in cilia cause a range of diseases. Vertebrate cilia respond to intercellular signaling molecules such as Hedgehog, a regulator of embryonic development. Despite its important roles in embryogenesis and disease, little is known about the evolutionary origins of ciliary signaling. Understanding how cilia evolved mechanisms to receive and transduce signals will help identify core ciliary functions fundamental to animal biology. To shed light on the evolution of ciliary signaling, I will characterize the signaling function of cilia in a close relative of animals, choanoflagellates. Choanoflagellates provide a simple biological system in which to elucidate mechanisms of ciliary signaling and identify signaling molecules that may be conserved in mammals. I will address two questions: (1) What signaling proteins are present in choanoflagellate cilia? And (2) what are the functions of choanoflagellate homologs of Hedgehog pathway components? I will isolate cilia from choanoflagellates to identify putative ciliary signaling proteins using mass spectrometry and carry out follow-up studies on candidate proteins prioritized based upon their homology to animal proteins. To understand the ancestry of a known ciliary signaling pathway, I will characterize choanoflagellate homologs of the Hedgehog pathway members Patched (receptor) and Hedgehog (ligand) using a heterologous expression system. This research will provide novel insights into the evolution of ciliary signaling and mechanisms of disease-associated signal transduction.

Intraflagellar Transport

Cell Signaling

10.1186/2046-2530-1-s1-p11

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Citations (0)

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

Nature (2008)

Nicole King M. Jody Westbrook Susan L. Young Alan Kuo Monika Abedin

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans. The genome sequence of the marine choanoflagellate Monosiga brevicollis has now been determined. Choanoflagellates are a mainly sessile group of protozoa resembling the 'feeding cells' of sponges, and are considered to be the closest living unicellular relatives of multicellular animals. Comparison of the M. brevicollis sequence with metazoan genomes suggests that the last unicellular ancestor of animals had intron-rich genes, some encoding protein domains characteristically associated with cell adhesion and the extracellular matrix in animals. This organism is strictly unicellular, but other choanoflagellates form colonies and may provide clues as to the origin of cell signalling and other systems in early metazoans.

Multicellular organism

ENCODE

10.1038/nature06617

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Citations (1,106)

Starting and Maintaining Monosiga brevicollis Cultures

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

INTRODUCTION Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. Most, if not all, choanoflagellates are heterotrophs that prey on bacteria. Thus, all choanoflagellate media provide nutrition for bacteria, which are in turn consumed by the choanoflagellates. If a culture is axenic or has low bacterial content, it can be supplemented with a specific bacterial strain that has been grown separately. On the other hand, if a culture already contains a well-established flora of bacteria, it can be cocultured with the choanoflagellate isolate. In either case, it is common to enrich the medium with an organic extract, such as liver extract, cereal grass infusion, or a mixture of proteose peptone and yeast extract. This protocol describes how to start cultures of the marine choanoflagellate species, Monosiga brevicollis , from frozen stocks. These cultures can then be maintained and expanded in preparation for DNA or RNA isolation or cell biological assays.

Axenic

Isolation

10.1101/pdb.prot5148

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Citations (28)

The Choanoflagellates: Heterotrophic Nanoflagellates and Sister Group of the Metazoa

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

INTRODUCTION Choanoflagellates are the closest living relatives of the metazoa, and the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. These heterotrophic flagellates are a cosmopolitan group of small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Monosiga brevicollis , a marine species, has emerged as a representative of the group because it is easily grown and manipulated in the laboratory and was the subject of a recently completed genome project. Because of the similarity in morphology and ecology shown across the order Choanoflagellida, the protocols mentioned in this article should be transferable to most choanoflagellate species.

Sister group

10.1101/pdb.emo116

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Citations (23)

Separation of Choanoflagellate and Bacterial Genomic DNA

Cold Spring Harbor Protocols (2009)

Nicole King Susan L. Young Monika Abedin Martin Carr B. S. C. Leadbeater

INTRODUCTION Choanoflagellates are heterotrophic nanoflagellates: small, colorless protozoa that are present in marine and freshwater environments as well as in hydrated soils. Because they are the closest living relatives of the metazoa, the study of their cell biology and genomes promises to provide new insights into metazoan ancestry and origins. However, the preparation of genomic DNA from cultures of Monosiga brevicollis or other choanoflagellates can be complicated by the presence of abundant bacterial DNA. Indeed, bacterial DNA can represent as much as 90% of the total yield from a standard genomic DNA preparation. This protocol describes the use of a CsCl gradient to separate choanoflagellate genomic DNA from the DNA of its bacterial prey. This strategy works only when the G+C content of the choanoflagellate genomic DNA is significantly different from that of its bacterial food. The final DNA sample from this protocol is expected to be 90%-95% enriched for choanoflagellate genomic DNA and to provide an adequate template for genome sequencing projects.

genomic DNA

Bacterial genome size

10.1101/pdb.prot5154

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Citations (7)