scholarly journals PACRG, a protein linked to ciliary motility, mediates cellular signaling

2016 ◽  
Vol 27 (13) ◽  
pp. 2133-2144 ◽  
Author(s):  
Catrina M. Loucks ◽  
Nathan J. Bialas ◽  
Martijn P. J. Dekkers ◽  
Denise S. Walker ◽  
Laura J. Grundy ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Cellular Signaling ◽  
Cell Types ◽  
Small Subset ◽  
Protein Signaling ◽  
Sensory Physiology ◽  
Ciliary Motility ◽  
C Elegans ◽  
Sensory Signaling ◽  
Associated Proteins

Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon–associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan.

scholarly journals xbx-4, a homolog of the Joubert syndrome gene FAM149B1, acts via the CCRK and MAK kinase cascade to regulate cilia morphology

2021 ◽  
Author(s):  
Ashish K Maurya ◽  
Piali Sengupta
Keyword(s):  
Sensory Neuron ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Cell Types ◽  
Joubert Syndrome ◽  
C Elegans ◽  
Kinase Cascade ◽  
Multiple Cell ◽  
Kinase Pathway ◽  
Sensory Functions

Primary cilia are microtubule (MT)-based organelles that mediate sensory functions in multiple cell types. Disruption of cilia structure or function leads to a diverse collection of diseases termed ciliopathies. Mutations in the DUF3719 domain-containing protein FAM149B1 have recently been shown to elongate cilia via unknown mechanisms and result in the ciliopathy Joubert syndrome. The highly conserved CCRK and MAK/RCK kinases negatively regulate cilia length and structure in Chlamydomonas, C. elegans, and mammalian cells. How the activity of this kinase cascade is tuned to precisely regulate cilia architecture is unclear. Here we identify XBX-4, a DUF3719 domain-containing protein related to human FAM149B1, as a novel regulator of the DYF-18 CCRK and DYF-5 MAK kinase pathway in C. elegans. As in dyf-18 and dyf-5 mutants, sensory neuron cilia are elongated in xbx-4 mutants and exhibit altered axonemal MT stability. XBX-4 promotes DYF-18 CCRK activity to regulate DYF-5 MAK function and localization. We find that Joubert syndrome-associated mutations in the XBX-4 DUF3719 domain also elongate cilia in C. elegans. Our results identify a new metazoan-specific regulator of this highly conserved kinase pathway, and suggest that FAM149B1 may similarly act via the CCRK/MAK kinase pathway to regulate ciliary homeostasis in humans.

scholarly journals Soft, dynamic hydrogel confinement improves kidney organoid lumen morphology and reduces epithelial-mesenchymal transition in culture

2021 ◽  
Author(s):  
Floor A.A. Ruiter ◽  
Francis L.C. Morgan ◽  
Nadia Roumans ◽  
Anika Schumacher ◽  
Gisela G. Slaats ◽  
...  
Keyword(s):  
Rational Design ◽  
Primary Cilia ◽  
Epithelial Mesenchymal Transition ◽  
Cell Types ◽  
Target Cells ◽  
Structure Property ◽  
Mesenchymal Transition ◽  
Cilia Formation ◽  
Associated Proteins ◽  
Kidney Organoids

Pluripotent stem cell-derived kidney organoids offer a promising solution to renal failure, yet current organoid protocols often lead to off-target cells and phenotypic alterations, preventing maturity. Here, we created various dynamic hydrogel architectures, conferring a controlled and biomimetic environment for organoid encapsulation. We investigated how hydrogel stiffness and stress relaxation affect renal phenotype and undesired fibrotic markers. We observed stiff hydrogel encapsulation led to an absence of certain renal cell types and signs of an epithelial-mesenchymal transition (EMT), whereas encapsulation in soft-stress-relaxing hydrogels led to all major renal segments, fewer fibrosis/EMT associated proteins, apical proximal tubule enrichment, and primary cilia formation, representing a significant improvement over current approaches to culture kidney organoids. Our findings show that engineering hydrogel mechanics and dynamics have a decided benefit for organoid culture. These structure-property-function relationships can enable rational design of materials, bringing us closer to functional engraftments and disease-modelling applications.

scholarly journals Forkhead transcription factor FKH-8 is a master regulator of primary cilia in C. elegans

2021 ◽  
Author(s):  
Rebeca Brocal-Ruiz ◽  
Ainara Esteve-Serrano ◽  
Carlos Mora-Martinez ◽  
Peter Swoboda ◽  
Juan Tena ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Genetic Diseases ◽  
Ciliated Cell ◽  
Cell Types ◽  
Forkhead Transcription Factor ◽  
Master Regulator ◽  
C Elegans ◽  
Direct Activation ◽  
Wide Range ◽  
Motile Cilia

SUMMARYCilia, either motile or non-motile (a.k.a primary or sensory), are complex evolutionary conserved eukaryotic structures composed of hundreds of proteins required for their assembly, structure and function that are collectively known as the ciliome. Ciliome mutations underlie a group of pleiotropic genetic diseases known as ciliopathies. Proper cilium function requires the tight coregulation of ciliome gene transcription, which is only fragmentarily understood. RFX transcription factors (TF) have an evolutionarily conserved role in the direct activation of ciliome genes both in motile and non-motile cilia cell types. In vertebrates, FoxJ1 and FoxN4 Forkhead (FKH) TFs work with RFX in the direct activation of ciliome genes, exclusively in motile cilia cell-types. No additional TFs have been described to act together with RFX in primary cilia cell-types in any organism. Here we describe FKH-8, a FKH TF, as master regulator of the primary ciliome in Caenorhabditis elegans. fkh-8 is expressed in all ciliated neurons in C. elegans, binds the regulatory regions of ciliome genes, regulates ciliome gene expression, cilium morphology and a wide range of behaviours mediated by sensory cilia. Importantly, we find FKH-8 function can be replaced by mouse FOXJ1 and FOXN4 but not by members of other mouse FKH subfamilies. In conclusion, our results show that RFX and FKH TF families act as master regulators of ciliogenesis also in sensory ciliated cell types and suggest that this regulatory logic could be an ancient trait predating functional cilia sub-specialization.

A Deficiency Screen for Zygotic Loci Required for Establishment and Patterning of the Epidermis in Caenorhabditis elegans

Genetics ◽  
1997 ◽  
Vol 146 (1) ◽  
pp. 185-206 ◽  
Author(s):  
Rebecca M Terns ◽  
Peggy Kroll-Conner ◽  
Jiangwen Zhu ◽  
Sooyoun Chung ◽  
Joel H Rothman
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Types ◽  
Epidermal Cells ◽  
Epidermal Differentiation ◽  
Apparent Effect ◽  
Internal Organs ◽  
C Elegans ◽  
Seam Cell ◽  
Genomic Regions ◽  
Caenorhabditis Elegans Genome

To identify genomic regions required for establishment and patterning of the epidermis, we screened 58 deficiencies that collectively delete at least ∼67% of the Caenorhabditis elegans genome. The epidermal pattern of deficiency homozygous embryos was analyzed by examining expression of a marker specific for one of the three major epidermal cell types, the seam cells. The organization of the epidermis and internal organs was also analyzed using a monoclonal antibody specific for epithelial adherens junctions. While seven deficiencies had no apparent effect on seam cell production, 21 were found to result in subnormal, and five in excess numbers of these cells. An additional 23 deficiencies blocked expression of the seam cell marker, in some cases without preventing cell proliferation. Two deficiencies result in multinucleate seam cells. Deficiencies were also identified that result in subnormal numbers of epidermal cells, hyperfusion of epidermal cells into a large syncytium, or aberrant epidermal differentiation. Finally, analysis of internal epithelia revealed deficiencies that cause defects in formation of internal organs, including circularization of the intestine and bifurcation of the pharynx lumen. This study reveals that many regions of the C. elegans genome are required zygotically for patterning of the epidermis and other epithelia.

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

scholarly journals Centrin2 regulates CP110 removal in primary cilium formation

2015 ◽  
Vol 208 (6) ◽  
pp. 693-701 ◽  
Author(s):  
Suzanna L. Prosser ◽  
Ciaran G. Morrison
Keyword(s):  
Calcium Binding ◽  
Primary Cilia ◽  
Reverse Genetics ◽  
Cell Types ◽  
Serum Starvation ◽  
Basal Bodies ◽  
Cilium Formation ◽  
Cellular Quiescence ◽  
Retinal Pigmented Epithelial Cells

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.

scholarly journals Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

2016 ◽  
Vol 27 (22) ◽  
pp. 3616-3626 ◽  
Author(s):  
Tanumoy Saha ◽  
Isabel Rathmann ◽  
Abhiyan Viplav ◽  
Sadhana Panzade ◽  
Isabell Begemann ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Growth Dynamics ◽  
Automated Analysis ◽  
Cell Types ◽  
Control Mechanisms ◽  
Spatially Resolved ◽  
Novel Approach ◽  
Associated Proteins

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia

2019 ◽  
Vol 509 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Sarina Han ◽  
Ko Miyoshi ◽  
Sho Shikada ◽  
Genki Amano ◽  
Yinshengzhuoma Wang ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Associated Proteins

scholarly journals The retromer complex regulates C. elegans development and mammalian ciliogenesis

2021 ◽  
Author(s):  
Shuwei Xie ◽  
Ellie Smith ◽  
Carter Dierlam ◽  
Danita Mathew ◽  
Angelina Davis ◽  
...  
Keyword(s):  
Potential Function ◽  
Primary Cilium ◽  
Mammalian Cells ◽  
Primary Cilia ◽  
Vulval Development ◽  
Sorting Nexin ◽  
Capping Protein ◽  
C Elegans ◽  
Retromer Complex ◽  
Mother Centriole

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, and suggest a novel role for the retromer in CP110 removal from the mother centriole.

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