Proteomic analysis of multiple primary cilia reveals a novel mode of ciliary development in mammals

Summary Cilia are structurally and functionally diverse organelles, whose malfunction leads to ciliopathies. While recent studies have uncovered common ciliary transport mechanisms, limited information is available on the proteome of cilia, particularly that of sensory subtypes, which could provide insight into their functional and developmental diversities. In the present study, we performed proteomic analysis of unique, multiple 9+0 cilia in choroid plexus epithelial cells (CPECs). The analysis of juvenile swine CPEC cilia identified 868 proteins. Among them, 396 were shared with the proteome of 9+0 photoreceptor cilia (outer segment), whereas only 152 were shared with the proteome of 9+2 cilia and flagella. Various signaling molecules were enriched in a CPEC-specific ciliome subset, implicating multiplicity of sensory functions. The ciliome also included molecules for ciliary motility such as Rsph9. In CPECs from juvenile swine or adult mouse, Rsph9 was localized to a subpopulation of cilia, whereas they were non-motile. Live imaging of mouse choroid plexus revealed that neonatal CPEC cilia could beat vigorously, and the motility waned and was lost within 1–2 weeks. The beating characteristics of neonatal CPEC cilia were variable and different from those of typical 9+2 cilia of ependyma, yet an Efhc1-mediated mechanism to regulate the beating frequency was shared in both types of cilia. Notably, ultrastructural analysis revealed the presence of not only 9+0 but also 9+2 and atypical ciliary subtypes in neonatal CPEC. Overall, these results identified both conserved and variable components of sensory cilia, and demonstrated a novel mode of ciliary development in mammals.

[1]  T. Stearns,et al.  Molecular characterization of centriole assembly in ciliated epithelial cells , 2007, The Journal of cell biology.

[2]  T. Tsumoto,et al.  Efhc1 deficiency causes spontaneous myoclonus and increased seizure susceptibility. , 2009, Human molecular genetics.

[3]  J. García-Verdugo,et al.  Adult Ependymal Cells Are Postmitotic and Are Derived from Radial Glial Cells during Embryogenesis , 2005, The Journal of Neuroscience.

[4]  Tanya M. Teslovich,et al.  Comparative Genomics Identifies a Flagellar and Basal Body Proteome that Includes the BBS5 Human Disease Gene , 2004, Cell.

[5]  K. Gull,et al.  Flagellar motility is required for the viability of the bloodstream trypanosome , 2006, Nature.

[6]  W. Marshall,et al.  Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  F. Delsuc.  Comparative Genomics , 2010, Lecture Notes in Computer Science.

[8]  J. Yates,et al.  Proteomic Analysis of Mammalian Primary Cilia , 2012, Current Biology.

[9]  M. Chilvers,et al.  Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods , 2000, Thorax.

[10]  Colin A. Johnson,et al.  Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities. , 2009, American journal of human genetics.

[11]  R. Gesteland,et al.  Changes in morphology and physiology of olfactory receptor cilia during development , 1982, Neuroscience.

[12]  Keith Gull,et al.  Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells , 2006, Journal of Cell Science.

[13]  T. Uemura,et al.  Lack of cadherins Celsr2 and Celsr3 impairs ependymal ciliogenesis, leading to fatal hydrocephalus , 2010, Nature Neuroscience.

[14]  L. Ostrowski,et al.  Investigation of the possible role of a novel gene, DPCD, in primary ciliary dyskinesia. , 2004, American journal of respiratory cell and molecular biology.

[15]  M. Mann,et al.  Proteomic characterization of the human centrosome by protein correlation profiling , 2003, Nature.

[16]  S. Sorokin.  Reconstructions of centriole formation and ciliogenesis in mammalian lungs. , 1968, Journal of cell science.

[17]  K. Narita,et al.  Multiple primary cilia modulate the fluid transcytosis in choroid plexus epithelium , 2009, Neuroscience Research.

[18]  S. Tovar,et al.  Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status , 2009, The Journal of physiology.

[19]  J. Beisson,et al.  Basal body/centriole assembly and continuity. , 2003, Current opinion in cell biology.

[20]  A. Herrero,et al.  R062: Immunohistochemical Expression of Annexin A1 in Normal and Chronically Inflamed Nasal Mucosa , 2003, Archives of otolaryngology--head & neck surgery.

[21]  P. Satir,et al.  Overview of structure and function of mammalian cilia. , 2007, Annual review of physiology.

[22]  N. Hirokawa,et al.  Left-Right Asymmetry and Kinesin Superfamily Protein KIF3A: New Insights in Determination of Laterality and Mesoderm Induction by kif3A− /− Mice Analysis , 1999, The Journal of cell biology.

[23]  J. Szmydynger-Chodobska,et al.  Choroid plexus: Target for polypeptides and site of their synthesis , 2001, Microscopy research and technique.

[24]  Z. Priel,et al.  Ciliary motility in brain ventricular system and trachea of hamster , 1985, Brain Research.

[25]  Edward N Pugh,et al.  The Proteome of the Mouse Photoreceptor Sensory Cilium Complex*S , 2007, Molecular & Cellular Proteomics.

[26]  Colin A. Johnson,et al.  A meckelin-filamin A interaction mediates ciliogenesis. , 2012, Human molecular genetics.

[27]  S. Suarez,et al.  Annexins Are Candidate Oviductal Receptors for Bovine Sperm Surface Proteins and Thus May Serve to Hold Bovine Sperm in the Oviductal Reservoir1 , 2007, Biology of reproduction.

[28]  L. Lim,et al.  The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. Pazour,et al.  Proteomic analysis of a eukaryotic cilium , 2005, The Journal of cell biology.

[30]  Gary Ruvkun,et al.  Analysis of xbx genes in C. elegans , 2005, Development.

[31]  Keith A. Boroevich,et al.  Functional Genomics of the Cilium, a Sensory Organelle , 2005, Current Biology.

[32]  Shankar Subramaniam,et al.  Decoding Cilia Function Defining Specialized Genes Required for Compartmentalized Cilia Biogenesis , 2004, Cell.

[33]  K. Mikoshiba,et al.  Lateral Diffusion of Inositol 1,4,5-Trisphosphate Receptor Type 1 Is Regulated by Actin Filaments and 4.1N in Neuronal Dendrites* , 2004, Journal of Biological Chemistry.

[34]  C. Madhavi,et al.  Atypical cilia in the choroid plexus of guineapig. , 1989, The Indian journal of medical research.

[35]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[36]  K. Sawamoto,et al.  Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia , 2010, Nature Cell Biology.

[37]  B. Engelhardt,et al.  C-C chemokine receptor 6–regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE , 2009, Nature Immunology.

[38]  M. Nachury,et al.  Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? , 2010, Annual review of cell and developmental biology.

[39]  T. Ideker,et al.  Functional genomic screen for modulators of ciliogenesis and cilium length , 2010, Nature.

[40]  S. Palay,et al.  The Fine Structure of the Nervous System: Neurons and Their Supporting Cells , 1991 .

[41]  N. Katsanis,et al.  The Vertebrate Primary Cilium in Development, Homeostasis, and Disease , 2009, Cell.

[42]  K. Narita,et al.  Structure and function of vertebrate cilia, towards a new taxonomy. , 2012, Differentiation; research in biological diversity.

[43]  S. Frings,et al.  The proteome of rat olfactory sensory cilia , 2009, Proteomics.

[44]  M. Mann,et al.  Parts per Million Mass Accuracy on an Orbitrap Mass Spectrometer via Lock Mass Injection into a C-trap*S , 2005, Molecular & Cellular Proteomics.

[45]  K. Yamakawa,et al.  Sequential expression of Efhc1/myoclonin1 in choroid plexus and ependymal cell cilia. , 2008, Biochemical and biophysical research communications.

[46]  N. Hirokawa,et al.  Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow. , 2009, Cold Spring Harbor perspectives in biology.

[47]  J. Yates,et al.  Proteomic Analysis of Isolated Chlamydomonas Centrioles Reveals Orthologs of Ciliary-Disease Genes , 2005, Current Biology.